Recombinant microorganisms for the production of fatty amines

ABSTRACT

The disclosure relates to recombinant microorganisms for the production of fatty amines and derivatives thereof. Further contemplated are cultured recombinant host cells as well as methods of producing fatty amines by employing these host cells.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a 371(c) national stage entry of PCT/US2014/068950,filed Dec. 5, 2014, which claims the benefit of U.S. ProvisionalApplication No. 61/912,184 filed Dec. 5, 2013, the contents of which arehereby incorporated-by-reference in their entireties.

FIELD

The disclosure relates to recombinant microorganisms for the productionof fatty amines and derivatives thereof. Further contemplated arerecombinant host cells that express biosynthetic proteins that convertfatty aldehydes to fatty amines in vivo. Still encompassed are methodsof producing fatty amines by employing the host cells expressing thesebiosynthetic proteins.

BACKGROUND

Fatty amines are nitrogen derivatives of fatty acids, olefins, oralcohols. They are made from natural fats and oils or from synthetic orpetrochemical raw materials. Today, these compounds are producedprimarily through the chemical modification of triglycerides such astallow or vegetable oils (e.g., coconut-, canola-, and rapeseed oil).

Commercially available fatty amines are made of either a mixture ofcarbon chains or a specific chain length that ranges from C₈ to C₂₂. Ingeneral, they are classified into primary-, secondary-, and tertiaryamines, depending on the number of hydrogen atoms of an ammonia moleculereplaced by fatty alkyl or methyl groups. Fatty amines are known to becationic surface-active compounds that strongly adhere to surfacesthrough either physical or chemical bonding. Many commercial productsare prepared using fatty amines as reactive intermediates. For example,they are useful as surfactants and as components of personal careproducts such as shampoos and conditioners. The largest market for fattyamines is in fabric softeners and detergents. Fatty amines are also usedas foaming- and wetting agents, antistatic agent in the textile andplastics industry, lubricants, paint thickeners, oil field chemicals,asphalt emulsifiers, petroleum additives, corrosion inhibitors,gasoline- and fuel oil additives, flotation agents, pigment wettingagents, epoxy curing agents, herbicides, and others (see Visek K. (2003)Fatty Amines; Kirk-Othmer Encyclopedia of Chemical Technology).

Producing fatty amines via microbial fermentation provides a number ofadvantages, such as providing a more consistent composition,manufacturing at a lower cost, and reducing the environmental impact. Inaddition, it would open up diverse new feedstocks that are beyond thenatural fats and oils and synthetic or petrochemical raw materials usedtoday. There is currently no efficient method for microbial productionof fatty amines. The disclosure addresses this need.

SUMMARY

One aspect of the disclosure provides a recombinant microorganism forthe production of a fatty amine, including an engineered metabolicpathway for converting a fatty aldehyde to a fatty amine. Herein, therecombinant microorganism has an engineered metabolic pathway forconverting a fatty aldehyde to a fatty amine that includes an exogenousbiosynthetic enzyme that has aminotransferase or amine dehydrogenaseactivity. In one embodiment, the exogenous biosynthetic enzyme is aputrescine aminotransferase such as YgjG. In another embodiment, theexogenous biosynthetic enzyme is a GABA aminotransferase such as PuuE.YgjG is encoded by a nucleic acid sequence that codes for an ygjG genethat is expressed in the recombinant microorganism or recombinantmicrobial cell. Similarly, PuuE is encoded by a nucleic acid sequencethat codes for a puuE gene that is expressed in the recombinantmicroorganism or recombinant microbial cell. In another embodiment, theexogenous biosynthetic enzyme is an amine dehydrogenase such asamethylamine dehydrogenase from Paracoccus denitrificans. Therecombinant microorganism or recombinant microbial cell produces thefatty amine in vivo or inside the cell. The fatty amine is released intoa culture medium by the recombinant microorganism or recombinantmicrobial cell. In one embodiment, the recombinant microorganism ormicrobial cell is a recombinant bacterial cell.

Another aspect of the disclosure provides a recombinant microorganismfor the production of a fatty amine, including a first engineeredmetabolic pathway for converting a fatty aldehyde to a fatty amine. Therecombinant microorganism has a first engineered metabolic pathway forconverting a fatty aldehyde to a fatty amine that includes an exogenousbiosynthetic enzyme that has aminotransferase or amine dehydrogenaseactivity (supra). In one embodiment, the recombinant microorganism hasanother or second engineered metabolic pathway for converting anacyl-ACP or an acyl-CoA to a fatty acid. The second engineered metabolicpathway is optional. Herein, the acyl-ACP or acyl-CoA is converted to afatty acid by a biosynthetic enzyme having thioesterase activity. In oneembodiment, the biosynthetic enzyme having thioesterase activity isencoded by a nucleic acid sequence that codes for a tesA gene that isexpressed in the recombinant microorganism or microbial cell. Therecombinant microorganism or recombinant microbial cell produces thefatty amine in vivo or inside the cell. The fatty amine is released intoa culture medium by the recombinant microorganism or recombinantmicrobial cell. In one embodiment, the recombinant microorganism ormicrobial cell is a recombinant bacterial cell.

Another aspect of the disclosure provides a recombinant microorganismfor the production of a fatty amine, including a first engineeredmetabolic pathway for converting a fatty aldehyde to a fatty amine. Therecombinant microorganism has a first engineered metabolic pathway forconverting a fatty aldehyde to a fatty amine that includes an exogenousbiosynthetic enzyme that has aminotransferase or amine dehydrogenaseactivity (supra). In one embodiment, the recombinant microorganism hasanother or second engineered metabolic pathway for converting anacyl-ACP or an acyl-CoA to a fatty acid (supra). In another embodiment,the recombinant microorganism has yet another or third engineeredmetabolic pathway for converting a fatty acid to a fatty aldehyde. Thisthird engineered metabolic pathway is optional and independent of thesecond engineered metabolic pathway. Herein, the fatty acid is convertedto a fatty aldehyde by a biosynthetic enzyme having carboxylic acidreductase (CAR) activity. In one embodiment, the biosynthetic enzymehaving CAR activity is encoded by a nucleic acid sequence that codes fora carB gene that is expressed in the recombinant microorganism ormicrobial cell. The recombinant microorganism or recombinant microbialcell produces the fatty amine in vivo or inside the cell. The fattyamine is released into a culture medium by the recombinant microorganismor recombinant microbial cell. In one embodiment, the recombinantmicroorganism or microbial cell is a recombinant bacterial cell.

Another aspect of the disclosure provides a recombinant bacterial cellfor production of a fatty amine, including one or more expressed genesthat encode an exogenous biosynthetic enzyme having thioesteraseactivity; one or more expressed genes that encode an exogenousbiosynthetic enzyme having carboxylic acid reductase activity; and oneor more expressed genes that encode an exogenous biosynthetic enzymehaving aminotransferase or amine dehydrogenase activity, wherein therecombinant bacterial cell produces a fatty amine in vivo or inside thebacterial cell. The fatty amine is released into a culture medium by therecombinant bacterial cell. Herein, the exogenous biosynthetic enzymehaving thioesterase activity converts an acyl-ACP or acyl-CoA to a fattyacid. The exogenous biosynthetic enzyme having carboxylic acid reductase(CAR) activity converts that fatty acid to a fatty aldehyde. Theexogenous biosynthetic enzyme having aminotransferase or aminedehydrogenase activity converts that fatty aldehyde to a fatty amine. Inone embodiment, the exogenous biosynthetic enzyme having thioesteraseactivity is encoded by a nucleic acid sequence that codes for a tesAgene. In another embodiment, the exogenous biosynthetic enzyme havingCAR activity is encoded by a nucleic acid sequence that codes for a carBgene. In still another embodiment, the exogenous biosynthetic enzymehaving aminotransferase activity is a putrescine aminotransferase suchas YgjG or a GABA aminotransferase such as PuuE. YgjG is encoded by anucleic acid sequence that codes for a ygjG gene. PuuE is encoded by anucleic acid sequence that codes for a puuE gene. In yet anotherembodiment, the exogenous biosynthetic enzyme having amine dehydrogenaseactivity is a methylamine dehydrogenase such as a methylaminedehydrogenase from Paracoccus denitrificans.

Another aspect of the disclosure provides a recombinant bacterial cellfor production of a fatty amine, including one or more expressed genesthat encode an exogenous biosynthetic enzyme having thioesteraseactivity to convert an acyl-ACP or an acyl-CoA to a fatty acid; one ormore expressed genes that encode an exogenous biosynthetic enzyme havingcarboxylic acid reductase (CAR) activity to convert the fatty acid to afatty aldehyde; and one or more expressed genes that encode an exogenousbiosynthetic enzyme having aminotransferase or amine dehydrogenaseactivity to convert the fatty aldehyde to a fatty amine, wherein therecombinant bacterial cell produces the fatty amine in vivo or insidethe cell. In one embodiment, the exogenous biosynthetic enzyme havingaminotransferase or amine dehydrogenase activity is a putrescineaminotransferase such as YgjG. In another embodiment, the exogenousbiosynthetic enzyme having aminotransferase or amine dehydrogenaseactivity is a GABA aminotransferase such as PuuE. YgjG is encoded by anucleic acid sequence that codes for an ygjG gene that is expressed inthe recombinant bacterial cell. Similarly, PuuE is encoded by a nucleicacid sequence that codes for a puuE gene that is expressed in therecombinant bacterial cell. In another embodiment, the exogenousbiosynthetic enzyme having aminotransferase or amine dehydrogenaseactivity is an amine dehydrogenase such as amethylamine dehydrogenase ofParacoccus denitrificans. In one embodiment, the exogenous biosyntheticenzyme having thioesterase activity is encoded by a nucleic acidsequence that codes for a tesA gene. In another embodiment, theexogenous biosynthetic enzyme having CAR activity is encoded by anucleic acid sequence that codes for a carB gene. The recombinantbacterial cell produces the fatty amine in vivo or inside the cell. Thefatty amine is released into a culture medium by the recombinantbacterial cell.

The disclosure further contemplates a recombinant bacterial cell for theproduction of a fatty amine, including a first engineered pathway forconverting an acyl-ACP or acyl-CoA to a fatty acid; a second engineeredmetabolic pathway for converting the fatty acid to a fatty aldehyde; anda third engineered metabolic pathway for converting the fatty aldehydeto a fatty amine, wherein the recombinant bacterial cell produces thefatty amine in vivo or inside the cell. In one embodiment, the acyl-ACPor acyl-CoA is converted to a fatty acid by an exogenously expressedbiosynthetic enzyme having thioesterase activity; the fatty acid isconverted to a fatty aldehyde by an exogenously expressed biosyntheticenzyme having carboxylic acid reductase (CAR) activity; and the fattyaldehyde is converted to a fatty amine by an exogenously expressedbiosynthetic enzyme having aminotransferase or amine dehydrogenaseactivity. In one embodiment, the exogenous biosynthetic enzyme havingaminotransferase or amine dehydrogenase activity is a putrescineaminotransferase such as YgjG. In another embodiment, the exogenousbiosynthetic enzyme having aminotransferase or amine dehydrogenaseactivity is a GABA aminotransferase such as PuuE. YgjG is encoded by anucleic acid sequence that codes for an ygjG gene that is expressed inthe recombinant bacterial cell. Similarly, PuuE is encoded by a nucleicacid sequence that codes for a puuE gene that is expressed in therecombinant bacterial cell. In another embodiment, the exogenousbiosynthetic enzyme having aminotransferase or amine dehydrogenaseactivity is an amine dehydrogenase such as a methylamine dehydrogenasefrom Paracoccus denitrificans. In one embodiment, the exogenousbiosynthetic enzyme having thioesterase activity is encoded by a nucleicacid sequence that codes for a tesA gene. In another embodiment, theexogenous biosynthetic enzyme having CAR activity is encoded by anucleic acid sequence that codes for a carB gene. The recombinantbacterial cell produces the fatty amine in vivo or inside the cell. Thefatty amine is released into a culture medium by the recombinantbacterial cell.

Another aspect of the disclosure provides a recombinant microorganismfor the production of a fatty amine, including but not limited to,Escherichia, Bacillus, Cyanophyta, Lactobacillus, Zymomonas,Rhodococcus, Pseudomonas, Aspergillus, Trichoderma, Neurospora,Fusarium, Humicola, Rhizomucor, Kluyveromyces, Pichia, Mucor,Myceliophtora, Penicillium, Phanerochaete, Pleurotus, Trametes,Chrysosporium, Saccharomyces, Stenotrophamonas, Schizosaccharomyces,Yarrowia, and Streptomyces. In one embodiment, Escherichia isEscherichia coli. In another embodiment, Cyanophyta includes, but is notlimited to, Prochlorococcus, Synechococcus, Synechocystis, Cyanothece,and Nostoc punctiforme. In still another embodiment, Cyanophytaincludes, but is not limited to, Synechococcus elongatus PCC7942,Synechocystis sp. PCC6803, and Synechococcus sp. PCC7001.

Another aspect of the disclosure provides a method of producing a fattyamine, comprising culturing a recombinant microorganism in afermentation broth containing a carbon source. The microorganismencompasses at least one engineered metabolic pathway for producing afatty amine in vivo (supra). Another aspect of the disclosure provides amethod of producing a fatty amine in a recombinant bacterial cell,including culturing a cell that expresses an engineered metabolicpathway for producing a fatty amine (supra) in a fermentation broth inthe presence of a carbon source; and harvesting fatty amines thatcollect in the fermentation broth.

The disclosure further encompasses a cell culture including arecombinant microbial cell for the production of amines (supra). In oneembodiment, the cell culture encompasses a recombinant bacterial cellfor the production of amines. In another embodiment, the recombinantmicrobial cell is a recombinant bacterial cell.

Another aspect of the disclosure provides a recombinant microorganismthat has an engineered metabolic pathway for fatty aldehyde productionand a biosynthetic enzyme that converts a fatty aldehyde to a fattyamine, wherein the biosynthetic enzyme has aminotransferase/transaminaseor amine dehydrogenase activity. In one embodiment, the biosyntheticenzyme is a putrescine aminotransferase or a GABA aminotransferase. Inanother embodiment, the biosynthetic enzyme is an amine dehydrogenase oran amine oxidase.

Another aspect of the disclosure provides a recombinant microorganismthat has an engineered metabolic pathway for fatty aldehyde productionand an engineered metabolic pathway for fatty amine production includinga biosynthetic enzyme that converts a fatty aldehyde to a fatty amine,wherein the recombinant microorganism is a microbial cell. In oneaspect, the microbial cell is a recombinant cell. The microbial cellincludes, but is not limited to, Escherichia, Bacillus, Cyanophyta,Lactobacillus, Zymomonas, Rhodococcus, Pseudomonas, Aspergillus,Trichoderma, Neurospora, Fusarium, Humicola, Rhizomucor, Kluyveromyces,Pichia, Mucor, Myceliophtora, Penicillium, Phanerochaete, Pleurotus,Trametes, Chrysosporium, Saccharomyces, Stenotrophamonas,Schizosaccharomyces, Yarrowia, and Streptomyces. In one embodiment,Escherichia is Escherichia coli. In another embodiment, Cyanophytaincludes, but is not limited to, Prochlorococcus, Synechococcus,Synechocystis, Cyanothece, and Nostoc punctiforme. In another particularembodiment, Cyanophyta is Synechococcus elongatus PCC7942, Synechocystissp. PCC6803, or Synechococcus sp. PCC7001.

Another aspect of the disclosure provides a recombinant microorganismthat has an engineered metabolic pathway for fatty aldehyde productionand an engineered metabolic pathway for fatty amine production. In oneembodiment, the engineered metabolic pathway for fatty aldehydeproduction includes a thioesterase and/or a carboxylic acid reductase(CAR) that convert a fatty acid to a fatty aldehyde, while theengineered metabolic pathway for fatty amine production includes anaminotransferase/transaminase or amine dehydrogenase that converts afatty aldehyde to a fatty amine. In some embodiments, the thioesteraseis encoded by a nucleic acid sequence that codes for a tesA gene with orwithout leader sequence while the carboxylic acid reductase (CAR) isencoded by a nucleic acid sequence that codes for a carB gene both ofwhich are expressed in the microorganism. In one embodiment, theaminotransferase/transaminase is a putrescine aminotransferase or a GABAaminotransferase. In one embodiment, the putrescine aminotransferase isYgjG which is encoded by a nucleic acid sequence that codes for an ygjGgene that is expressed in the microorganism. In another embodiment, theGABA aminotransferase is PuuE which is encoded by a nucleic acidsequence that codes for a puuE gene that is expressed in themicroorganism. In still another embodiment, the amine dehydrogenase is amethylamine dehydrogenase from Paracoccus denitrificans. In oneembodiment, the fatty amine is released into the supernatant or culturemedia by the microorganism. In another embodiment, the fatty amine iscollected from inside the microorganism where it can be extracted duringor after a fermentation procedure.

The disclosure further contemplates a method of producing a fatty amine,including culturing a recombinant microorganism in a fermentation brothcontaining a carbon source, wherein the recombinant microorganismcontains an engineered metabolic pathway for fatty aldehyde productionand a biosynthetic enzyme that converts a fatty aldehyde to a fattyamine, wherein the biosynthetic enzyme has aminotransferase or aminedehydrogenase activity, and wherein the microorganism produces a fattyamine in vivo.

The disclosure further encompasses a recombinant microbial cell thatincludes expression of one or more enzymes having thioesterase andcarboxylic acid reductase (CAR) activity; and expression of an enzymehaving aminotransferase or amine dehydrogenase activity, wherein themicrobial cell produces fatty amines. In one embodiment, thebiosynthetic enzyme is a putrescine aminotransferase such as YgjG or aGABA aminotransferase such as PuuE. In another embodiment, thebiosynthetic enzyme is an amine dehydrogenase such as a methylaminedehydrogenase from Paracoccus denitrificans. In yet another embodiment,the biosynthetic enzyme is an amine oxidase.

Still, another aspect of the disclosure provides a method for producinga fatty amine in a recombinant microorganism. The method includesculturing a microbial cell (supra) in a fermentation medium in thepresence of a carbon source; and harvesting the fatty amines thatcollect in the supernatant or fermentation medium. The recombinantmicrobial cell expresses an engineered metabolic pathway for fattyaldehyde production and a biosynthetic enzyme that converts a fattyaldehyde to a fatty amine, wherein the biosynthetic enzyme hasaminotransferase or amine dehydrogenase activity. In another aspect, therecombinant microbial cell expresses an engineered metabolic pathway forfatty aldehyde production and an engineered metabolic pathway for amineproduction including a biosynthetic enzyme that converts a fattyaldehyde to a fatty amine, wherein the biosynthetic enzyme hasaminotransferase or amine dehydrogenase activity.

Another aspect of the disclosure provides a fatty amine derived from acarbon source that is not a petrochemical raw material. For example, thedisclosure provides for fatty amines derived from renewable feedstocks,such as CO₂, CO, glucose, sucrose, xylose, arabinose, glycerol, mannose,or mixtures thereof. Other feedstocks provided herein from which fattyamines may be derived include starches, cellulosic biomass, molasses,and other sources of carbohydrates including carbohydrate mixturesderived from hydrolysis of cellulosic biomass, or the waste materialsderived from plant- or natural oil processing.

BRIEF DESCRIPTION OF THE FIGURES

The present disclosure is best understood when read in conjunction withthe accompanying figures, which serve to illustrate the preferredembodiments. It is understood, however, that the disclosure is notlimited to the specific embodiments disclosed in the figures.

FIG. 1 is a MS/GC chromatograph from microbial cell extracts showing aunique fatty amine peak (see middle panel at 7.61 minutes, marked withan arrow) produced via expression of a thioesterase, a carboxylic acidreductase, and an aminotransferase/transaminase (middle row). The toprow is the F16 negative control; the middle row is the F16-YG sample;and the bottom row is the YG negative control. Additional peaks in thetop and middle rows are fatty alcohols.

FIG. 2 depicts another MS/GC chromatograph showing the elution profileof the F16-YG sample (top row) as compared to the 1-dodecylaminereference standard (bottom row). Additional peaks in the top and middlerows are fatty alcohols.

FIG. 3 shows the ion fragmentation pattern of the 7.6 minutes peak fromthe F16-YG sample (top row) and the 1-dodecylamine reference standard(bottom row). The molecular structure of characteristic ion fragments of1-dodecylamine, including C₁₁H₂₄N, C₁₀H₂₂N, C₉H₂₀N, are also shown inthe top row.

DETAILED DESCRIPTION General Overview

The disclosure relates to microbial production of fatty amines, whichrepresent a new class of renewable chemical products. The fatty aminesare produced through a microorganism that expresses an engineeredmetabolic pathway to convert fatty aldehydes to fatty amines. As such,the microorganism expresses at least one exogenous biosynthetic enzymein order to produce fatty amines in vivo. The exogenous biosyntheticenzyme may have aminotransferase or amine dehydrogenase activity; orcarboxylic acid reductase (CAR) activity; or thioesterase activity or acombination thereof. Alternative forms of the enzymatic activity arealso encompassed herein.

Definitions

As used in this specification and the appended claims, the singularforms “a,” “an” and “the” include plural referents unless the contextclearly dictates otherwise. Thus, for example, reference to “a hostcell” includes two or more such host cells, reference to “a fatty amine”includes one or more fatty amines, or mixtures of amines, reference to“a nucleic acid sequence” includes one or more nucleic acid sequences,reference to “an enzyme” includes one or more enzymes, and the like.

The term “engineered metabolic pathway” refers to one or moregenetically engineered or optimized chemical reaction(s) catalyzed by atleast one biosynthetic enzyme expressed in a cell in order to produce(or increase production of) a certain substance (i.e., a precursor, anintermediate or an end product) inside that cell. In one embodiment, thebiosynthetic enzyme is an exogenous biosynthetic enzyme. In anotherembodiment, the engineered metabolic pathway permits the cell'sproduction of a desired end product. In another embodiment, theengineered metabolic pathway permits the cell's production of a desiredprecursor. In another embodiment, the engineered metabolic pathwaypermits the cell's production of a desired intermediate.

The term “in vivo” refers to “inside the cell” when used in context ofproducing a specific product. For example, production of fatty amines invivo means production of fatty amines inside the cell.

Sequence Accession numbers as referred to herein were obtained fromdatabases provided by the NCBI (National Center for BiotechnologyInformation) maintained by the National Institutes of Health, U.S.A.(which are identified herein as “NCBI Accession Numbers” oralternatively as “GenBank Accession Numbers”), and from the UniProtKnowledgebase (UniProtKB) and Swiss-Prot databases provided by the SwissInstitute of Bioinformatics (which are identified herein as “UniProtKBAccession Numbers”).

Enzyme Classification (EC) Numbers are established by the NomenclatureCommittee of the International Union of Biochemistry and MolecularBiology (IUBMB), a description of which is available on the IUBMB EnzymeNomenclature website on the World Wide Web. EC numbers classify enzymesaccording to the enzyme-catalyzed reactions. For example, if differentenzymes (e.g., from different organisms) catalyze the same reaction,then they are classified under the same EC number. In addition, throughconvergent evolution, different protein folds can catalyze identicalreactions and therefore are assigned identical EC numbers (seeOmelchenko et al. (2010) Biol. Direct 5:31). Proteins that areevolutionarily unrelated and can catalyze the same biochemical reactionsare sometimes referred to as analogous enzymes (i.e., as opposed tohomologous enzymes). EC numbers differ from, for example, UniProtidentifiers which specify a protein by its amino acid sequence.

As used herein, the term “nucleotide” refers to a monomeric unit of apolynucleotide that consists of a heterocyclic base, a sugar, and one ormore phosphate groups. The naturally occurring bases (guanine, (G),adenine, (A), cytosine, (C), thymine, (T), and uracil (U)) are typicallyderivatives of purine or pyrimidine, though it should be understood thatnaturally and non-naturally occurring base analogs are also included.The naturally occurring sugar is the pentose (five-carbon sugar)deoxyribose (which forms DNA) or ribose (which forms RNA), though itshould be understood that naturally and non-naturally occurring sugaranalogs are also included. Nucleic acids are typically linked viaphosphate bonds to form nucleic acids or polynucleotides, though manyother linkages are known in the art (e.g., phosphorothioates,boranophosphates, and the like).

The term “polynucleotide” refers to a polymer of ribonucleotides (RNA)or deoxyribonucleotides (DNA), which can be single-stranded ordouble-stranded and which can contain non-natural or alterednucleotides. The terms “polynucleotide,” “nucleic acid sequence,” and“nucleotide sequence” are used interchangeably herein and refer to apolymeric form of nucleotides of any length. These terms refer to theprimary structure of the molecule, and thus include double- andsingle-stranded DNA, and double- and single-stranded RNA. The termsinclude, as equivalents, analogs of either RNA or DNA made fromnucleotide analogs and modified polynucleotides such as, though notlimited to methylated and/or capped polynucleotides. The polynucleotidecan be in any form, including but not limited to, plasmid, viral,chromosomal, EST, cDNA, mRNA, and rRNA.

As used herein, the terms “polypeptide” and “protein” are usedinterchangeably to refer to a polymer of amino acid residues. The term“recombinant polypeptide” refers to a polypeptide that is produced byrecombinant techniques, wherein generally cDNA or RNA encoding theexpressed protein is inserted into a suitable expression vector that isin turn used to transform a host cell to produce the polypeptide.Similarly, the terms “recombinant polynucleotide” or “recombinantnucleic acid” or “recombinant DNA” are produced by recombinanttechniques that are known to those of skill in the art.

As used herein, the terms “homolog,” and “homologous” refer to apolynucleotide or a polypeptide comprising a sequence that is at leastabout 50 percent (%) identical to the corresponding polynucleotide orpolypeptide sequence. Preferably homologous polynucleotides orpolypeptides have polynucleotide sequences or amino acid sequences thathave at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least about 99%homology to the corresponding amino acid sequence or polynucleotidesequence. As used herein the terms sequence “homology” and “sequenceidentity” are used interchangeably.

One of ordinary skill in the art is well aware of methods to determinehomology between two or more sequences. Briefly, calculations of“homology” between two sequences can be performed as follows. Thesequences are aligned for optimal comparison purposes (e.g., gaps can beintroduced in one or both of a first and a second amino acid or nucleicacid sequence for optimal alignment and non-homologous sequences can bedisregarded for comparison purposes). In one preferred embodiment, thelength of a first sequence that is aligned for comparison purposes is atleast about 30%, preferably at least about 40%, more preferably at leastabout 50%, even more preferably at least about 60%, and even morepreferably at least about 70%, at least about 80%, at least about 85%,at least about 90%, at least about 95%, at least about 98%, or about100% of the length of a second sequence. The amino acid residues ornucleotides at corresponding amino acid positions or nucleotidepositions of the first and second sequences are then compared. When aposition in the first sequence is occupied by the same amino acidresidue or nucleotide as the corresponding position in the secondsequence, then the molecules are identical at that position. The percenthomology between the two sequences is a function of the number ofidentical positions shared by the sequences, taking into account thenumber of gaps and the length of each gap, that need to be introducedfor optimal alignment of the two sequences. The comparison of sequencesand determination of percent homology between two sequences can beaccomplished using a mathematical algorithm, such as BLAST (Altschul etal. (1990) J. Mol. Biol. 215(3):403-410). The percent homology betweentwo amino acid sequences also can be determined using the Needleman andWunsch algorithm that has been incorporated into the GAP program in theGCG software package, using either a Blossum 62 matrix or a PAM250matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a lengthweight of 1, 2, 3, 4, 5, or 6 (Needleman and Wunsch (1970) J. Mol. Biol.48:444-453). The percent homology between two nucleotide sequences alsocan be determined using the GAP program in the GCG software package,using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80and a length weight of 1, 2, 3, 4, 5, or 6. One of ordinary skill in theart can perform initial homology calculations and adjust the algorithmparameters accordingly. A preferred set of parameters (and the one thatshould be used if a practitioner is uncertain about which parametersshould be applied to determine if a molecule is within a homologylimitation of the claims) are a Blossum 62 scoring matrix with a gappenalty of 12, a gap extend penalty of 4, and a frameshift gap penaltyof 5. Additional methods of sequence alignment are known in thebiotechnological arts (see, e.g., Rosenberg (2005) BMC Bioinformatics6:278; Altschul et al. (2005) FEBS J. 272(20):5101-5109).

The term “hybridizes under low stringency, medium stringency, highstringency, or very high stringency conditions” describes conditions forhybridization and washing. Guidance for performing hybridizationreactions can be found in biotechnological texts (e.g., see CurrentProtocols in Molecular Biology, John Wiley & Sons, N.Y. (1989),6.3.1-6.3.6, where aqueous and non-aqueous methods are described indetail and either method can be used). For example, specifichybridization conditions are as follows: (1) low stringencyhybridization conditions—6× sodium chloride/sodium citrate (SSC) atabout 45° C., followed by two washes in 0.2×SSC, 0.1% SDS at least at50° C. (the temperature of the washes can be increased to 55° C. for lowstringency conditions); (2) medium stringency hybridizationconditions—6×SSC at about 45° C., followed by one or more washes in0.2×SSC, 0.1% SDS at 60° C.; (3) high stringency hybridizationconditions—6×SSC at about 45° C., followed by one or more washes in0.2.×SSC, 0.1% SDS at 65° C.; and (4) very high stringency hybridizationconditions—0.5M sodium phosphate, 7% SDS at 65° C., followed by one ormore washes at 0.2×SSC, 1% SDS at 65° C. Very high stringency conditions(4) are generally the preferred conditions unless otherwise specified.

The term “endogenous” means “originating within”. As such, an“endogenous” polypeptide refers to a polypeptide that is encoded by thenative genome of the host cell. For example, an endogenous polypeptidecan refer to a polypeptide that is encoded by the genome of the parentalmicrobial cell (e.g., the parental host cell) from which the recombinantcell is engineered (or derived).

The term “exogenous” means “originating from outside”. As such, an“exogenous” polypeptide refers to a polypeptide which is not encoded bythe native genome of the cell. An exogenous polypeptide and/or exogenouspolynucleotide can be transferred into the cell and can be cloned fromor derived from a different cell type or species; or can be cloned fromor derived from the same cell type or species. For example, an“exogenous biosynthetic enzyme” is an example of an exogenouspolypeptide, wherein the polypeptide codes for an enzyme having acertain enzymatic activity. In another example, a variant (i.e., mutantor altered) polypeptide is an example of an exogenous polypeptide.Similarly, a non-naturally-occurring nucleic acid molecule is consideredto be exogenous to a cell once introduced into the cell. The term“exogenous” may also be used with reference to a polynucleotide,polypeptide, or protein which is present in a recombinant host cell in anon-native state. For example, an “exogenous” polynucleotide,polypeptide or protein sequence may be modified relative to the wildtype sequence naturally present in the corresponding wild type hostcell, e.g., a modification in the level of expression or in the sequenceof a polynucleotide, polypeptide or protein. Along those same lines, anucleic acid molecule that is naturally-occurring can also be exogenousto a particular cell. For example, an entire coding sequence isolatedfrom cell X is an exogenous nucleic acid with respect to cell Y oncethat coding sequence is introduced into cell Y, even if X and Y are thesame cell type.

The term “overexpressed” means that a gene is caused to be transcribedat an elevated rate compared to the endogenous transcription rate forthat gene. In some examples, overexpression additionally includes anelevated rate of translation of the corresponding protein compared tothe endogenous translation rate for that protein. In some embodiments,the term “overexpress” means to express a polynucleotide or polypeptidein a cell at a greater concentration than is normally expressed in acorresponding wild-type cell under the same conditions. Methods oftesting for overexpression are well known in the art, for exampletranscribed RNA levels can be assessed using rtPCR and protein levelscan be assessed using SDS page gel analysis.

The term “heterologous” means “derived from a different organism,different cell type, and/or different species”. As used herein, the term“heterologous” is typically associated with a polynucleotide or apolypeptide or a protein and refers to a polynucleotide, a polypeptideor a protein that is not naturally present in a given organism, celltype, or species. For example, a polynucleotide sequence from a plantcan be introduced into a microbial host cell by recombinant methods, andthe plant polynucleotide is then heterologous to that recombinantmicrobial host cell. Similarly, a polynucleotide sequence fromcyanobacteria can be introduced into a microbial host cell of the genusEscherichia by recombinant methods, and the polynucleotide fromcyanobacteria is then heterologous to that recombinant microbial hostcell. Along those lines, a “heterologous biosynthetic enzyme” is anexample of a heterologous polypeptide, wherein the polypeptide codes foran enzyme having a certain enzymatic activity.

As used herein, the term “fragment” of a polypeptide refers to a shorterportion of a full-length polypeptide or protein ranging in size from twoamino acid residues to the entire amino acid sequence minus one aminoacid residue. In certain embodiments of the disclosure, a fragmentrefers to the entire amino acid sequence of a domain of a polypeptide orprotein (e.g., a substrate binding domain or a catalytic domain).

The term “mutagenesis” refers to a process by which the geneticinformation of an organism is changed in a stable manner. Mutagenesis ofa protein coding nucleic acid sequence produces a mutant protein.Mutagenesis also refers to changes in non-coding nucleic acid sequencesthat result in modified protein activity.

A “mutation”, as used herein, refers to a permanent change in a nucleicacid position of a gene or in an amino acid position of a polypeptide orprotein. Mutations include substitutions, additions, insertions, and/ordeletions. For example, a mutation in an amino acid position can be asubstitution of one type of amino acid with another type of amino acid(e.g., a serine (S) may be substituted with an alanine (A); a lysine (L)may be substituted with an T (Threonine); etc.). As such, a polypeptideor a protein can have one or more mutations wherein one amino acid issubstituted with another amino acid. For example, a biosyntheticpolypeptide or protein can have one or more mutations in its amino acidsequence.

The term “biosynthetic enzyme” as used herein, refers to a protein thathas an enzymatic activity that is related to fatty acid derivativebiosynthesis (e.g., fatty acids, fatty aldehydes, fatty alcohols, fattyamines, fatty esters, etc.). An example of a biosynthetic enzyme as usedherein is an enzyme that can convert a fatty aldehyde precursor to afatty amine (e.g., a fatty amine producing biosynthetic enzyme). Anotherexample of a biosynthetic enzyme as used herein is an enzyme that canconvert a fatty acid to a fatty aldehyde (e.g., a fatty aldehydeproducing biosynthetic enzyme). Still another example of a biosyntheticenzyme as used herein is an enzyme that can convert an acyl-ACP oracyl-CoA to a fatty acid (e.g., a fatty acid producing biosyntheticenzyme). When a cell has been transformed with a biosynthetic enzyme itis a cell that expresses the biosynthetic enzyme (e.g., a recombinantcell). In one embodiment, the titer and/or yield of a fatty aminerelated compound produced by a cell that expresses a fatty amineproducing biosynthetic enzyme is at least twice that of a correspondingwild type cell (i.e., a corresponding cell that does not express thefatty amine producing biosynthetic enzyme). In another embodiment, thetiter and/or yield of a fatty amine related compound produced by a cellthat expresses the fatty amine producing biosynthetic enzyme is at leastabout 1 times, at least about 2 times, at least about 3 times, at leastabout 4 times, at least about 5 times, at least about 6 times, at leastabout 7 times, at least about 8 times, at least about 9 times, or atleast about 10 times greater than that of a corresponding wild typecell. In one embodiment, the titer and/or yield of a fatty amine relatedcompound produced by a cell expressing a fatty amine producingbiosynthetic enzyme is at least about 1 percent, at least about 2percent, at least about 3 percent, at least about 4 percent, at leastabout 5 percent, at least about 6 percent, at least about 7 percent, atleast about 8 percent, at least about 9 percent, or about 10 percentgreater than that of a corresponding wild type cell. In anotherembodiment, the titer and/or yield due to the expression of a fattyamine producing biosynthetic enzyme is at least about 20 percent to atleast about 100 percent greater than that of the wild type cell. Inanother embodiments, the titer and/or yield of a fatty amine relatedcompound produced by a cell due to the expression of a fatty amineproducing biosynthetic enzyme is at least about 20 percent, at leastabout 25 percent, at least about 30 percent, at least about 35 percent,at least about 40 percent, at least about 45 percent at least about 50percent, at least about 55 percent, at least about 60 percent, at leastabout 65 percent, at least about 70 percent, at least about 75 percent,at least about 80 percent, at least about 85 percent, at least about 90percent, at least about 95 percent, at least about 97 percent, at leastabout 98 percent, or at least about 100 percent greater than that of thecorresponding wild type cell.

As used herein, the term “gene” refers to nucleic acid sequencesencoding either an RNA product or a protein product, as well asoperably-linked nucleic acid sequences affecting the expression of theRNA or protein (e.g., such sequences include but are not limited topromoter or enhancer sequences) or operably-linked nucleic acidsequences encoding sequences that affect the expression of the RNA orprotein (e.g., such sequences include but are not limited to ribosomebinding sites or translational control sequences).

Expression control sequences are known in the art and include, forexample, promoters, enhancers, polyadenylation signals, transcriptionterminators, internal ribosome entry sites (IRES), and the like, thatprovide for the expression of the polynucleotide sequence in a hostcell. Expression control sequences interact specifically with cellularproteins involved in transcription (Maniatis et al., Science, 236:1237-1245 (1987)). Exemplary expression control sequences are describedin biotechnological texts (e.g., see Goeddel, Gene ExpressionTechnology: Methods in Enzymology, Vol. 185, Academic Press, San Diego,Calif. (1990)). In the methods of the present disclosure, one or moreexpression control sequences are operably linked to one or morepolynucleotide sequences. By “operably linked” is meant that apolynucleotide sequence and an expression control sequence are connectedin such a way as to permit gene expression when the appropriatemolecules (e.g., transcriptional activator proteins) are bound to theexpression control sequence. Operably linked promoters are locatedupstream of the selected polynucleotide sequence in terms of thedirection of transcription and translation. Operably linked enhancerscan be located upstream, within, or downstream of the selectedpolynucleotide.

As used herein, the term “vector” refers to a nucleic acid moleculecapable of transporting another nucleic acid, i.e., a polynucleotidesequence, to which it has been linked. One type of useful vector is anepisome (i.e., a nucleic acid capable of extra-chromosomal replication).Useful vectors are those capable of autonomous replication and/orexpression of nucleic acids to which they are linked. Vectors capable ofdirecting the expression of genes to which they are operatively linkedare referred to herein as “expression vectors.” In general, expressionvectors of utility in recombinant DNA techniques are often in the formof “plasmids,” which refer generally to circular double stranded DNAloops that, in their vector form, are not bound to the chromosome. Otheruseful expression vectors are provided in linear form. Also included aresuch other forms of expression vectors that serve equivalent functionsand that have become known in the art subsequently hereto. In someembodiments, a recombinant vector further includes a promoter operablylinked to the polynucleotide sequence. In some embodiments, the promoteris a developmentally-regulated promoter, an organelle-specific promoter,a tissue-specific promoter, an inducible promoter, a constitutivepromoter, or a cell-specific promoter. The recombinant vector typicallycomprises at least one sequence selected from an expression controlsequence operatively coupled to the polynucleotide sequence; a selectionmarker operatively coupled to the polynucleotide sequence; a markersequence operatively coupled to the polynucleotide sequence; apurification moiety operatively coupled to the polynucleotide sequence;a secretion sequence operatively coupled to the polynucleotide sequence;and a targeting sequence operatively coupled to the polynucleotidesequence. In certain embodiments, the nucleotide sequence is stablyincorporated into the genomic DNA of the host cell, and the expressionof the nucleotide sequence is under the control of a regulated promoterregion. The expression vectors as used herein include a particularpolynucleotide sequence as described herein in a form suitable forexpression of the polynucleotide sequence in a host cell. It will beappreciated by those skilled in the art that the design of theexpression vector can depend on such factors as the choice of the hostcell to be transformed, the level of expression of polypeptide desired,etc. The expression vectors described herein can be introduced into hostcells to produce polypeptides, including fusion polypeptides, encoded bythe polynucleotide sequences as described herein.

The terms “recombinant cell” and “recombinant host cell” are usedinterchangeably herein and refer to a cell that has been modified toexogenously express at least one biosynthetic enzyme. In one particularembodiment, the biosynthetic enzyme can convert a fatty aldehydeprecursor into a fatty amine. Thus, in one embodiment, the recombinantcell encompasses a biosynthetic enzyme that can increase the specificactivity of the recombinant cell to produce fatty amines or fatty aminederived compounds. A recombinant cell can be derived from amicroorganism or microbial cell such as a bacterium, a virus or afungus. The recombinant cell can be used to produce fatty amines. Insome embodiments, the recombinant cell exogenously expresses one or morepolynucleotide(s), each polynucleotide encoding a polypeptide havingbiosynthetic enzyme activity, wherein the recombinant cell produces afatty amine related composition when cultured in the presence of acarbon source under conditions effective to express thepolynucleotide(s).

As used herein, the term “microorganism” refers to a microscopicorganism. Examples of a microorganism are a bacterium, a virus, or afungus. In one embodiment, a microorganism is a bacterial cell. Inanother embodiment, a microorganism is a prokaryote or prokaryotic cell.In yet another embodiment, a microorganism is a fungal cell such as ayeast cell. In another embodiment, a microorganism is a viral cell. In arelated embodiment, a “recombinant microorganism” is a microorganismthat has been genetically altered and expresses or encompasses anexogenous and/or heterologous nucleic acid sequence. In another relatedembodiment, a “recombinant microorganism” is a microorganism that hasbeen genetically altered and expresses an engineered metabolic pathwaythat includes at least one exogenously expressed protein (e.g., anexogenous biosynthetic enzyme).

The term “acyl-ACP” refers to an acyl thioester formed between thecarbonyl carbon of the alkyl chain and the sulfhydryl group of thephosphopantetheinyl moiety of an acyl carrier protein (ACP). Thephosphopantetheinyl moiety is post-translationally attached to aconserved serine residue on the ACP by the action of holo-acyl carrierprotein synthase (ACPS), a phosphopantetheinyl transferase. In someembodiments an acyl-ACP is an intermediate in the synthesis of fullysaturated acyl-ACPs. In other embodiments an acyl-ACP is an intermediatein the synthesis of unsaturated acyl-ACPs. In some embodiments, thecarbon chain will have about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 carbons. Each of theseacyl-ACPs are substrates for enzymes that convert them to fatty acidderivatives.

The term “acyl-CoA” is a temporary compound formed when coenzyme A (CoA)attaches to the end of a fatty acid inside a living cell. It refers to agroup of coenzymes that are involved in the metabolism of fatty acids.In some embodiments, the carbon chain will have about 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26carbons. Each of these acyl-CoAs are substrates for enzymes that convertthem to fatty acid derivatives.

The term “metabolic pathway for fatty aldehyde production” means anybiosynthetic pathway that produces fatty aldehydes. The metabolicpathway for fatty aldehyde production may include any number of enzymesto produce fatty aldehydes.

The term “metabolic pathway for fatty amine production” means anybiosynthetic pathway that produces fatty amines. The metabolic pathwayfor fatty amine production may include at least one enzyme to producefatty amines.

As used herein, “fatty amine” means an amine having the formula RNH2. Afatty amine as referred to herein can be any fatty amine made from, forexample, a fatty acid or fatty aldehyde or fatty aldehyde derived from afatty acyl-ACP. In some embodiments, the R group is at least 5, at least6, at least 7, at least 8, at least 9, at least 10, at least 11, atleast 12, at least 13, at least 14, at least 15, at least 16, at least17, at least 18, or at least 19 carbons in length. Alternatively, or inaddition, the R group is 24 or less, 23 or less, 22 or less, 20 or less,19 or less, 18 or less, 17 or less, 16 or less, 15 or less, 14 or less,13 or less, 12 or less, 11 or less, 10 or less, 9 or less, 8 or less, 7or less, or 6 or less carbons in length. Thus, the R group can have an Rgroup bounded by any two of the above endpoints. For example, the Rgroup can be 6-16 carbons in length, 10-14 carbons in length, or 12-18carbons in length. In some embodiments, the fatty amine compositioncomprises one or more of a C6, C7, C8, C9, C10, C11, C12, C13, C14, C15,C16, C17, C18, C19, C20, C21, 22, 23, and a C24 fatty amine. In otherembodiments, the fatty amine composition includes one or more of a C6,C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, and a C18 fattyamine. In still other embodiments, the fatty amine composition includesC12, C14, C16 and C18 fatty amines; C12, C14 and C16 fatty amines; C14,C16 and C18 fatty amines; or C12 and C14 fatty amines. The R group of afatty amine, can be a straight chain or a branched chain. Branchedchains may have more than one point of branching and may include cyclicbranches. In some embodiments, the branched fatty amine is a C6, C7, C8,C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, C20, C21, C22, C23or C24 branched fatty amine. The R group of a branched or unbranchedfatty amine can be saturated or unsaturated. If unsaturated, the R groupcan have one or more than one point of unsaturation. In someembodiments, the unsaturated fatty amine is a monounsaturated fattyamine. In certain embodiments, the unsaturated fatty amine is a C6:1,C7:1, C8:1, C9:1, C10:1, C11:1, C12:1, C13:1, C14:1, C15:1, C16:1,C17:1, C18:1, C19:1, C20:1, C21:1, C22:1, C23:1 or a C24:1 unsaturatedfatty amine. In certain embodiments, the unsaturated fatty amine is aC10:1, C12:1, C14:1, C16:1, or C18:1 unsaturated fatty amine. In otherembodiments, the unsaturated fatty amine is unsaturated at the omega-7position. In certain embodiments, the unsaturated fatty amine has a cisdouble bond. Fatty amines are classified into primary-, secondary-, andtertiary amines, depending on the number of hydrogen atoms of an ammoniamolecule replaced by fatty alkyl or methyl groups. Examples of fattyamines that can be used as detergents, in water treatments, as flotationagents, in petroleum, as corrosion inhibitors, in textiles, in rubber,and the like, are tertiary amines such as di-methyl alkyl amines (C10,C12, C14, C16 or C18) and di-alkyl methyl amines (C10); and tertiaryamine blends such as di-methyl alkyl amines (C8-C18, C12-C18, C12-C14,or C16-C18).

The term “clone” typically refers to a cell or group of cells descendedfrom and essentially genetically identical to a single common ancestor,for example, the bacteria of a cloned bacterial colony arose from asingle bacterial cell.

As used herein, the term “culture” typical refers to a liquid mediacomprising viable cells. In one embodiment, a culture comprises cellsreproducing in a predetermined culture media under controlledconditions, for example, a culture of recombinant host cells grown inliquid media comprising a selected carbon source and nitrogen.“Culturing” or “cultivation” refers to growing a population of hostcells (e.g., recombinant host cells) under suitable conditions in aliquid or solid medium. In some embodiments, culturing refers to thefermentative bioconversion of a substrate to an end-product. Culturingmedia are well known and individual components of such culture media areavailable from commercial sources (e.g., DIFCO media and BBL media). Inone example, the aqueous nutrient medium is a “rich medium” includingcomplex sources of nitrogen, salts, and carbon, such as YP medium,including 10 g/L of peptone and 10 g/L yeast extract. In anotherexample, the nutrient medium is a “minimal medium” composed of traceelements, nutrients, and salts.

The terms, “a modified activity” or “an altered level of activity”, forexample, with respect to an enzymatic activity in a recombinant hostcell, refers to a difference in one or more characteristics in theenzyme activity as determined relative to the parent or native hostcell. Typically, such differences in activity are determined between arecombinant host cell (i.e., having modified activity) and thecorresponding wild-type host cell, particularly by comparing the cultureof a recombinant host cell with the culture of the correspondingwild-type host cell. Modified activities can be the result of, forexample, modified amounts of protein expressed by a recombinant hostcell (e.g., as the result of increased or decreased number of copies ofDNA sequences encoding the protein, increased or decreased number ofmRNA transcripts encoding the protein, and/or increased or decreasedamounts of protein translation of the protein from mRNA); changes in thestructure of the protein (e.g., changes to the primary structure, suchas, changes to the protein's coding sequence that result in changes insubstrate specificity, changes in observed kinetic parameters); andchanges in protein stability (e.g., increased or decreased degradationof the protein). In certain instances, the coding sequences for thepolypeptides described herein are codon optimized for expression in aparticular host cell. For example, for expression in E. coli, one ormore codons can be optimized accordingly (see Grosjean et al. (1982)Gene 18:199-209).

The term “regulatory sequences” as used herein typically refers to asequence of bases in DNA, operably-linked to DNA sequences encoding aprotein that ultimately controls the expression of the protein. Examplesof regulatory sequences include, but are not limited to, RNA promotersequences, transcription factor binding sequences, transcriptiontermination sequences, modulators of transcription (such as enhancerelements), nucleotide sequences that affect RNA stability, andtranslational regulatory sequences (such as, ribosome binding sites(e.g., Shine-Dalgarno sequences in prokaryotes or Kozak sequences ineukaryotes), initiation codons, termination codons). As used herein, thephrase “the expression of said nucleotide sequence is modified relativeto the wild type nucleotide sequence,” means an increase or decrease inthe level of expression and/or activity of an endogenous nucleotidesequence or the expression and/or activity of a heterologous ornon-native polypeptide-encoding nucleotide sequence. The terms “alteredlevel of expression” and “modified level of expression” are usedinterchangeably and mean that a polynucleotide, polypeptide, orhydrocarbon is present in a different concentration in an engineeredhost cell as compared to its concentration in a corresponding wild-typecell under the same conditions. As used herein, the term “express” withrespect to a polynucleotide is to cause it to function. A polynucleotidewhich encodes a polypeptide (or protein) will, when expressed, betranscribed and translated to produce that polypeptide (or protein).

As used herein, the term “titer” refers to the quantity of a fatty amineor fatty amine related compound or composition produced per unit volumeof host cell culture. In any aspect of the compositions and methodsdescribed herein, a fatty amine is produced at a titer of about 25 mg/L,about 50 mg/L, about 75 mg/L, about 100 mg/L, about 125 mg/L, about 150mg/L, about 175 mg/L, about 200 mg/L, about 225 mg/L, about 250 mg/L,about 275 mg/L, about 300 mg/L, about 325 mg/L, about 350 mg/L, about375 mg/L, about 400 mg/L, about 425 mg/L, about 450 mg/L, about 475mg/L, about 500 mg/L, about 525 mg/L, about 550 mg/L, about 575 mg/L,about 600 mg/L, about 625 mg/L, about 650 mg/L, about 675 mg/L, about700 mg/L, about 725 mg/L, about 750 mg/L, about 775 mg/L, about 800mg/L, about 825 mg/L, about 850 mg/L, about 875 mg/L, about 900 mg/L,about 925 mg/L, about 950 mg/L, about 975 mg/L, about 1000 mg/L, about1050 mg/L, about 1075 mg/L, about 1100 mg/L, about 1125 mg/L, about 1150mg/L, about 1175 mg/L, about 1200 mg/L, about 1225 mg/L, about 1250mg/L, about 1275 mg/L, about 1300 mg/L, about 1325 mg/L, about 1350mg/L, about 1375 mg/L, about 1400 mg/L, about 1425 mg/L, about 1450mg/L, about 1475 mg/L, about 1500 mg/L, about 1525 mg/L, about 1550mg/L, about 1575 mg/L, about 1600 mg/L, about 1625 mg/L, about 1650mg/L, about 1675 mg/L, about 1700 mg/L, about 1725 mg/L, about 1750mg/L, about 1775 mg/L, about 1800 mg/L, about 1825 mg/L, about 1850mg/L, about 1875 mg/L, about 1900 mg/L, about 1925 mg/L, about 1950mg/L, about 1975 mg/L, about 2000 mg/L (2 g/L), 3 g/L, 5 g/L, 10 g/L, 20g/L, 30 g/L, 40 g/L, 50 g/L, 60 g/L, 70 g/L, 80 g/L, 90 g/L, 100 g/L ora range bounded by any two of the foregoing values. In otherembodiments, a fatty amine is produced at a titer of more than 100 g/L,more than 200 g/L, or more than 300 g/L. One preferred titer of fattyamine produced by a recombinant host cell according to the methods ofthe disclosure is from 5 g/L to 200 g/L, 10 g/L to 150 g/L, 20 g/L to120 g/L, and 30 g/L to 100 g/L. The titer may refer to a particularfatty amine or a combination or composition of fatty amines produced bya given recombinant host cell culture. For example, the expression ofbiosynthetic protein that can convert a fatty aldehyde to a fatty aminein a recombinant host cell such as E. coli results in the production ofa higher titer as compared to a recombinant host cell expressing thecorresponding wild type polypeptide that lacks the expression of thebiosynthetic protein that can convert a fatty aldehyde to a fatty amine.In one embodiment, the higher titer ranges from at least about 5 g/L toabout 200 g/L.

As used herein, the “yield of a fatty amine related compound includingfatty amines produced by a host cell” refers to the efficiency by whichan input carbon source is converted to product in a host cell. Hostcells engineered to produce a fatty amine or fatty amine relatedcompound according to the methods of the disclosure have a yield of atleast about 3%, at least about 4%, at least about 5%, at least about 6%,at least about 7%, at least about 8%, at least about 9%, at least about10%, at least about 11%, at least about 12%, at least about 13%, atleast about 14%, at least about 15%, at least about 16%, at least about17%, at least about 18%, at least about 19%, at least about 20%, atleast about 21%, at least about 22%, at least about 23%, at least about24%, at least about 25%, at least about 26%, at least about 27%, atleast about 28%, at least about 29%, or at least about 30% or a rangebounded by any two of the foregoing values. In other embodiments, afatty amine is produced at a yield of more than about 30%, more thanabout 35%, more than about 40%, more than about 45%, more than about50%, more than about 55%, more than about 60%, more than about 65%, morethan about 70%, more than about 75%, more than about 80%, more thanabout 85%, more than about 90% or higher. Alternatively, or in addition,the yield is about 30% or less, about 27% or less, about 25% or less, orabout 22% or less. In another embodiment, the yield is about 50% orless, about 45% or less, or about 35% or less. In another embodiment,the yield is about 95% or less, or 90% or less, or 85% or less, or 80%or less, or 75% or less, or 70% or less, or 65% or less, or 60% or less,or 55% or less, or 50% or less. Thus, the yield can be bounded by anytwo of the above endpoints. For example, the yield of a fatty amine orfatty amine related compound produced by the recombinant host cellaccording to the methods of the disclosure can be about 5% to about 15%,about 10% to about 25%, about 10% to about 22%, about 15% to about 27%,about 18% to about 22%, about 20% to about 28%, about 20% to about 30%,about 30% to about 40%, about 40% to about 50%, about 50% to about 60%,about 60% to about 70%, about 70% to about 80%, about 80% to about 90%,or about 90% to about 100%. The yield may refer to a particular fattyamine or fatty amine related compound or a combination of fatty aminesproduced by a given recombinant host cell culture. For example, theexpression of a biosynthetic protein that can convert a fatty aldehydeto a fatty amine in a recombinant host cell such as E. coli results inthe production of a higher yield of fatty amines or fatty amine derivedcompounds including compositions or blends of fatty amines as comparedto a host cell expressing the corresponding wild type polypeptide. Inone embodiment, the higher yield ranges from about 10% to about 100% oftheoretical yield.

As used herein, the term “productivity” refers to the quantity of afatty amine or fatty amine related compound including a composition orblend of one or more fatty amines produced per unit volume of host cellculture per unit time. In any aspect of the compositions and methodsdescribed herein, the productivity of a fatty amine related compoundincluding a composition or blend of fatty amines produced by arecombinant host cell is at least 100 mg/L/hour, at least 200 mg/L/hour,at least 300 mg/L/hour, at least 400 mg/L/hour, at least 500 mg/L/hour,at least 600 mg/L/hour, at least 700 mg/L/hour, at least 800 mg/L/hour,at least 900 mg/L/hour, at least 1000 mg/L/hour, at least 1100mg/L/hour, at least 1200 mg/L/hour, at least 1300 mg/L/hour, at least1400 mg/L/hour, at least 1500 mg/L/hour, at least 1600 mg/L/hour, atleast 1700 mg/L/hour, at least 1800 mg/L/hour, at least 1900 mg/L/hour,at least 2000 mg/L/hour, at least 2100 mg/L/hour, at least 2200mg/L/hour, at least 2300 mg/L/hour, at least 2400 mg/L/hour, 2500mg/L/hour, or as high as 10 g/L/hour (dependent upon cell mass). Forexample, the productivity of a fatty amine related compound including acomposition or blend of fatty amines produced by a recombinant host cellaccording to the methods of the present disclosure may be from 500mg/L/hour to 2500 mg/L/hour, or from 700 mg/L/hour to 2000 mg/L/hour.The productivity may refer to a particular fatty amine related compoundincluding a composition of fatty amines or a blend of fatty amine or acombination of fatty amines produced by a given host cell culture. Forexample, the expression of a biosynthetic protein that can convert afatty aldehyde to a fatty amine in a recombinant host cell such as E.coli results in the production of an increased productivity of fattyamine derived compounds including fatty amines and compositions andblends thereof as compared to a recombinant host cell expressing thecorresponding wild type polypeptide. In one embodiment, the higherproductivity ranges from about 0.3 g/L/h to about 3 g/L/h.

As used herein, the term “total fatty species” and “total fatty amineproduct” and “fatty amine derivative” may be used interchangeably hereinwith reference to the amount of fatty amines that can be produced by thehost cell that expresses the biosynthetic protein that can convert afatty aldehyde to a fatty amine, as evaluated by GC-FID.

As used herein, the term “glucose utilization rate” means the amount ofglucose used by the culture per unit time, reported as grams/liter/hour(g/L/hr).

As used herein, the term “carbon source” refers to a substrate orcompound suitable to be used as a source of carbon for prokaryotic orsimple eukaryotic cell growth. Carbon sources can be in various forms,including, but not limited to polymers, carbohydrates, acids, alcohols,aldehydes, ketones, amino acids, peptides, and gases (e.g., CO and CO₂).Exemplary carbon sources include, but are not limited to,monosaccharides, such as glucose, fructose, mannose, galactose, xylose,and arabinose; oligosaccharides, such as fructo-oligosaccharide andgalacto-oligosaccharide; polysaccharides such as starch, cellulose,pectin, and xylan; disaccharides, such as sucrose, maltose, cellobiose,and turanose; cellulosic material and variants such as hemicelluloses,methyl cellulose and sodium carboxymethyl cellulose; saturated orunsaturated fatty acids, succinate, lactate, and acetate; alcohols, suchas ethanol, methanol, and glycerol, or mixtures thereof. The carbonsource can also be a product of photosynthesis, such as glucose. Incertain embodiments, the carbon source is gas mixture containing COcoming from flu gas. In another embodiment, the carbon source is a gasmixture containing CO coming from the reformation of a carbon containingmaterial, such as biomass, coal, or natural gas. In other embodimentsthe carbon source is syngas, methane, or natural gas. In certainpreferred embodiments, the carbon source is biomass. In other preferredembodiments, the carbon source is glucose. In other preferredembodiments the carbon source is sucrose. In other embodiments thecarbon source is glycerol. In other preferred embodiments the carbonsource is sugar can juice, sugar cane syrup, or corn syrup. In otherpreferred embodiments, the carbon source is derived from renewablefeedstocks, such as CO₂, CO, glucose, sucrose, xylose, arabinose,glycerol, mannose, or mixtures thereof. In other embodiments, the carbonsource is derived from renewable feedstocks including starches,cellulosic biomass, molasses, and other sources of carbohydratesincluding carbohydrate mixtures derived from hydrolysis of cellulosicbiomass, or the waste materials derived from plant- or natural oilprocessing.

As used herein, the term “biomass” refers to any biological materialfrom which a carbon source is derived. In some embodiments, a biomass isprocessed into a carbon source, which is suitable for bioconversion. Inother embodiments, the biomass does not require further processing intoa carbon source. The carbon source can be converted into a compositioncomprising fatty amines. An exemplary source of biomass is plant matteror vegetation, such as corn, sugar cane, or switchgrass. Anotherexemplary source of biomass is metabolic waste products, such as animalmatter (e.g., cow manure). Further exemplary sources of biomass includealgae and other marine plants. Biomass also includes waste products fromindustry, agriculture, forestry, and households, including, but notlimited to, glycerol, fermentation waste, ensilage, straw, lumber,sewage, garbage, cellulosic urban waste, and food leftovers (e.g.,soaps, oils and fatty acids). The term “biomass” can also refer tosources of carbon, such as carbohydrates (e.g., monosaccharides,disaccharides, or polysaccharides).

As used herein, the term “isolated,” with respect to products (such asfatty amines and compositions and blends thereof) refers to productsthat are separated from cellular components, cell culture media, orchemical or synthetic precursors. The fatty amines produced by themethods described herein can be relatively immiscible in thefermentation broth, as well as in the cytoplasm. Therefore, the fattyamines can collect in an organic phase either intracellularly orextracellularly.

As used herein, the terms “purify,” “purified,” or “purification” meanthe removal or isolation of a molecule from its environment by, forexample, isolation or separation. “Substantially purified” molecules areat least about 60% free (e.g., at least about 70% free, at least about75% free, at least about 85% free, at least about 90% free, at leastabout 95% free, at least about 97% free, and at least about 99% free)from other components with which they are associated. As used herein,these terms also refer to the removal of contaminants from a sample. Forexample, the removal of contaminants can result in an increase in thepercentage of fatty amine derived compounds including fatty amines andcompositions and blends thereof in a sample. For example, when a fattyamine related compound is produced in a recombinant host cell, the fattyamine related compound can be purified by the removal of host cellproteins and/or other host cell material. After purification, thepercentage of fatty amines in the sample is increased. The terms“purify,” “purified,” and “purification” are relative terms which do notrequire absolute purity. Thus, for example, when a fatty amine relatedcompound is produced in recombinant host cells, the fatty amine compoundis substantially separated from other cellular components (e.g., nucleicacids, polypeptides, lipids, carbohydrates, or other hydrocarbons).

As used herein, the term “attenuate” means to weaken, reduce, ordiminish. For example, a polypeptide can be attenuated by modifying thepolypeptide to reduce its activity (e.g., by modifying a nucleotidesequence that encodes the polypeptide).

The term “fatty aldehyde producing biosynthetic enzyme” or “fattyaldehyde generating enzyme” are used interchangeably herein and refer toa polypeptide or a protein or an enzyme that has the enzymatic activityto generate fatty aldehydes or fatty aldehyde precursors. Examples ofsuch enzymes include, but are not limited to, a carboxylic acidreductase (CAR) (e.g., CarB) and/or a thioesterase (TE) (e.g., TesA,'tesA); an acyl-ACP reductase (AAR) (e.g., from Synechococcus elongatusPCC7942); an acyl-CoA reductase (e.g., Acr1); a phosphopanthetheinyltransferase (PPTase); and the like.

From Fatty Aldehyde Precursors to Fatty Amines

The disclosure relates to microbial production of fatty amines. As shownherein, microorganisms can be genetically engineered to express variousbiosynthetic enzymes in order to produce fatty amines in vivo. Morespecifically, a microorganism can be genetically engineered to express ametabolic pathway that converts a fatty aldehyde to a fatty amine. Thepathway expresses at least one biosynthetic enzyme that hasaminotransferase (e.g., putrescine aminotransferase (YgjG) or GABAaminotransferase (PuuE) from Escherichia coli) or amine dehydrogenase(e.g., methylamine dehydrogenase of Paracoccus denitrificans orquinohemo protein amine dehydrogenase of Pseudomonas spp.) or amineoxidase activity to convert fatty aldehydes to fatty amines.Transaminases carry out the same reaction as aminotransferases and thesenames are sometimes used interchangeably. For example, GABAaminotransferase is also referred to as 4-aminobutyrate transaminase. Inone embodiment, the aminotransferase or transaminase or aminedehydrogenase or amine oxidase is an exogenous biosynthetic enzyme orexogenously expressed in the cell. Alternatively, the fatty amines canbe produced by combining the engineered pathways for fatty amineproduction expressing a biosynthetic enzyme that has aminotransferase oramine dehydrogenase or amine oxidase activity to convert fatty aldehydesto fatty amines with an engineered pathway for fatty aldehyde productionexpressing an exogenous biosynthetic enzyme that produces fattyaldehydes. Such fatty aldehyde precursors can be generated in vivothrough a variety of processes and engineered pathways, such as anengineered carboxylic acid reductase (CAR) pathway utilized for fattyaldehyde production (see U.S. Pat. No. 8,097,439, incorporated herein byreference) or an engineered CAR and thioesterase (TE) pathway utilizedfor fatty alcohol production (see U.S. Pat. No. 8,097,439, supra) or anengineered phosphopanthetheinyl transferase (PPTase) and CAR pathwayutilized for fatty aldehyde or fatty alcohol production (see U.S.Application Publication No. 20130035513, incorporated herein byreference) or an engineered acyl-ACP reductase (AAR) pathway utilizedfor fatty aldehyde production (see U.S. Pat. No. 8,268,599, incorporatedherein by reference) or for alkane production (see U.S. Pat. No.8,323,924, incorporated herein by reference). An example of a suitableTE is ‘TesA (i.e., a truncated thioesterase from E. coli that has itsperiplasm leader sequence removed (hence the apostrophe), such that itremains in the cytoplasm); an example of a suitable CAR is CarB; anexample of a suitable AAR is the enzyme from Synechococcus elongatusPCC7942 (see Table 6, infra).

In one embodiment, co-expression of an engineered pathway for fattyaldehyde production and an engineered pathway for fatty amine productionwith the expression of an biosynthetic enzyme having aminotransferase oramine dehydrogenase or amine oxidase activity, along withsupplementation with an appropriate nitrogen source (e.g., glutamate orammonia or hydrogen peroxide) allows for the conversion of an aldehydeprecursor into the corresponding amine (see Table 1 for putrescineaminotransferase; see Tables 2, 3 and 4 for examples of enzymaticreactions). The availability of nitrogen donors (e.g., glutamate) forthe aminotransferase reaction can be optionally enhanced by overexpresssing glutamate dehydrogenase to increase the rate of glutamatebiosynthesis. The native E. coli glutamate dehydrogenase uses NADPH as aredox cofactor, thus, replacing this enzyme with a glutamatedehydrogenase that is capable of using NADH instead (such as, forexample, the glutamate dehydrogenases from Bacteroides spp. including,but not limited to, B. thetaiotaomicron, B. fragilis, B. distasonis, B.ovatus, B. vulgatus, and B. Uniformis) can increase the availability ofNADPH in the cell. This can provide an increased supply of NADPH forother biosynthetic processes that depend on NADPH (e.g., fatty acidbiosynthesis).

Table 1 below (infra) depicts EC numbers and names for variousbiosynthetic enzymes that are useful in the production of aminesincluding aminotransferases/transaminases. The enzyme commission orclassification number (EC number) is a numerical classification schemefor enzymes, based on the chemical reactions they catalyze. The ECnumbers do not technically specify enzymes, rather they specifyenzyme-catalyzed reactions. For example, if different enzymes (e.g.,from different organisms) catalyze the same reaction, then they receivethe same EC number. In addition, different protein folds can catalyze anidentical reaction and can therefore be assigned an identical EC numberbecause of convergent evolution (i.e., these are called non-homologousisofunctional enzymes, or NISE). As shown in Table 1, the enzymaticactivity of putrescine aminotransferase is classified under EC number2.6.1.82. All the enzymes shown in Table 1 participate in chemicalreactions that result in amines and are classified asaminotransferases/transaminases because their EC number falls under EC2.6.1 (see also Table 7, infra). Table 2 below (infra) shows a number ofdifferent biosynthetic enzymes that can produce fatty amines from fattyaldehyde precursors. For example, aminotransferases or transaminases,amine oxidases and amine dehydrogenases catalyze reactions where fattyaldehyde precursors are converted to fatty amines. In one embodiment, anengineered metabolic pathway includes an aminotransferase ortransaminase for production of fatty aldehydes in vivo. In anotherembodiment, an engineered metabolic pathway includes an amine oxidasefor production of fatty aldehydes in vivo. In another embodiment, anengineered metabolic pathway includes an amine dehydrogenase forproduction of fatty aldehydes in vivo. Table 3 below (infra) shows thereaction catalyzed by putrescine aminotransferase, i.e., convertingfatty aldehydes to fatty amines.

TABLE 1 Enzymes Involved in the Production of Amines under EC 2.6.12.6.1.1 Aspartate transaminase. 2.6.1.2 Alanine transaminase. 2.6.1.3Cysteine transaminase. 2.6.1.4 Glycine transaminase. 2.6.1.5 Tyrosinetransaminase. 2.6.1.6 Leucine transaminase. 2.6.1.7Kynurenine-oxoglutarate transaminase. 2.6.1.8 2,5-diaminovaleratetransaminase. 2.6.1.9 Histidinol-phosphate transaminase. 2.6.1.10Transferred entry: 2.6.1.21. 2.6.1.11 Acetylornithine transaminase.2.6.1.12 Alanine-oxo-acid transaminase. 2.6.1.13 Ornithineaminotransferase. 2.6.1.14 Asparagine-oxo-acid transaminase. 2.6.1.15Glutamine-pyruvate transaminase. 2.6.1.16 Glutamine-fructose-6-phosphatetransaminase (isomerizing). 2.6.1.17 Succinyldiaminopimelatetransaminase. 2.6.1.18 Beta-alanine-pyruvate transaminase. 2.6.1.194-aminobutyrate transaminase. 2.6.1.20 Deleted entry. 2.6.1.21D-amino-acid transaminase. 2.6.1.22 (S)-3-amino-2-methylpropionatetransaminase. 2.6.1.23 4-hydroxyglutamate transaminase. 2.6.1.24Diiodotyrosine transaminase. 2.6.1.25 Transferred entry: 2.6.1.24.2.6.1.26 Thyroid-hormone transaminase. 2.6.1.27 Tryptophan transaminase.2.6.1.28 Tryptophan-phenylpyruvate transaminase. 2.6.1.29 Diaminetransaminase. 2.6.1.30 Pyridoxamine-pyruvate transaminase. 2.6.1.31Pyridoxamine-oxaloacetate transaminase. 2.6.1.32Valine-3-methyl-2-oxovalerate transaminase. 2.6.1.33dTDP-4-amino-4,6-dideoxy-D-glucose transaminase. 2.6.1.34UDP-2-acetamido-4-amino-2,4,6-trideoxyglucose transaminase. 2.6.1.35Glycine-oxaloacetate transaminase. 2.6.1.36 L-lysine 6-transaminase.2.6.1.37 2-aminoethylphosphonate-pyruvate transaminase. 2.6.1.38Histidine transaminase. 2.6.1.39 2-aminoadipate transaminase. 2.6.1.40(R)-3-amino-2-methylpropionate-pyruvate transaminase. 2.6.1.41D-methionine-pyruvate transaminase. 2.6.1.42 Branched-chain-amino-acidtransaminase. 2.6.1.43 Aminolevulinate transaminase. 2.6.1.44Alanine-glyoxylate transaminase. 2.6.1.45 Serine-glyoxylatetransaminase. 2.6.1.46 Diaminobutyrate-pyruvate transaminase. 2.6.1.47Alanine-oxomalonate transaminase. 2.6.1.48 5-aminovalerate transaminase.2.6.1.49 Dihydroxyphenylalanine transaminase. 2.6.1.50Glutamine-scyllo-inositol transaminase. 2.6.1.51 Serine-pyruvatetransaminase. 2.6.1.52 Phosphoserine transaminase. 2.6.1.53 Transferredentry: 1.4.1.13. 2.6.1.54 Pyridoxamine-phosphate transaminase. 2.6.1.55Taurine-2-oxoglutarate transaminase. 2.6.1.561D-1-guanidino-3-amino-1,3-dideoxy-scyllo-inositol transaminase.2.6.1.57 Aromatic-amino-acid transaminase. 2.6.1.58Phenylalanine(histidine)transaminase. 2.6.1.59dTDP-4-amino-4,6-dideoxygalactose transaminase. 2.6.1.60Aromatic-amino-acid-glyoxylate transaminase. 2.6.1.61 Deleted entry.2.6.1.62 Adenosylmethionine-8-amino-7-oxononanoate transaminase.2.6.1.63 Kynurenine-glyoxylate transaminase. 2.6.1.64Glutamine-phenylpyruvate transaminase. 2.6.1.65 N(6)-acetyl-beta-lysinetransaminase. 2.6.1.66 Valine-pyruvate transaminase. 2.6.1.672-aminohexanoate transaminase. 2.6.1.68 Ornithine(lysine)transaminase.2.6.1.69 Deleted entry. 2.6.1.70 Aspartate-phenylpyruvate transaminase.2.6.1.71 Lysine-pyruvate 6-transaminase. 2.6.1.72D-4-hydroxyphenylglycine transaminase. 2.6.1.73 Methionine-glyoxylatetransaminase. 2.6.1.74 Cephalosporin-C-transaminase. 2.6.1.75Cysteine-conjugate transaminase. 2.6.1.76 Diaminobutyrate-2-oxoglutaratetransaminase. 2.6.1.77 Taurine-pyruvate aminotransferase. 2.6.1.78Aspartate-prephenate aminotransferase. 2.6.1.79 Glutamate-prephenateaminotransferase. 2.6.1.80 Nicotianamine aminotransferase. 2.6.1.81Succinylornithine transaminase.

2.6.1.83 LL-diaminopimelate aminotransferase. 2.6.1.84 Arginine-pyruvatetransaminase. 2.6.1.85 Aminodeoxychorismate synthase. 2.6.1.862-amino-4-deoxychorismate synthase.

TABLE 2 Enzymatic Reactions that Produce Fatty Amines Fatty AmineProduction from Fatty Aldehydes Enzymes ReactionsAminotransferase/transaminase

Amine oxidase

Amine dehydrogenase

TABLE 3 Enzymatic Reaction of Putrescine Aminotransferase PutrescineAminotransferase Enzymatic Reaction

In order to illustrate the disclosure, recombinant host cells wereengineered to express a thioesterase (TE), which catalyzes theconversion of acyl-ACPs or acyl-CoAs into free fatty acids; and acarboxylic acid reductase (CAR), which converts the free fatty acidsinto fatty aldehydes. The recombinant host cells were further engineeredto express a putrescine aminotransferase (YgjG) in order to convert thefatty aldehydes to fatty amines (see Example 1 for experimental results;see Table 3 (supra) for the enzymatic reaction carried out by YgjG; seeTable 5 (infra) for the aminotransferase/transaminase mechanism).Herein, the ygjG gene was cloned from E. coli and ligated into anexpression vector to generate an expression plasmid. A second expressionplasmid was generated by cloning and integrating a carB and a tesA gene.Cells were then transformed with both plasmids and cultured in afermentation broth with a carbon source. The production of fatty amineswas confirmed while control cells did not produce fatty amines (seeExample 1). Any suitable aminotransferase/transaminase can be used toproduce fatty amines so long as the enzymatic activity can convert fattyaldehydes into fatty amines. If the cell naturally produces fattyaldehydes then the cell is engineered to express an exogenous putrescineaminotransferase (YgjG) as shown in Example 1 in order to convert thenaturally present fatty aldehydes to fatty amines.

In another embodiment, an amine dehydrogenase can be used instead of anaminotransferase to convert fatty aldehydes into fatty amines (see Table4 (infra) for the enzymatic reaction carried out by an aminedehydrogenase; and Table 7 (infra) for examples of enzymes). This isapplicable if the nitrogen source is ammonia rather than amino acids.Examples of amine dehydrogenases are useful to convert fatty aldehydesto fatty amines fall under EC numbers EC 1.4.9; EC 1.4.98; and EC 1.4.99(see Table 7, infra). For example, alanine dehydrogenase, glutamatedehydrogenase, L-lysine-6-dehydrogenase; and methylamine dehydrogenaseare examples of amine dehydrogenases that can be used to convert fattyaldehydes into fatty amines (see Table 7, infra).

In yet another embodiment, amine oxidase can be used instead of anaminotransferase to convert fatty aldehydes into fatty amines.

TABLE 4 Amine Dehydrogenase Reaction Amine Dehydrogenase EnzymaticReaction

TABLE 5 Mechanism for Aminotransferase/Transaminase AminotransferaseMechanism

Tables 6 and 7 below (infra) depict various enzymatic activities andtheir corresponding enzyme classification (EC) numbers.

TABLE 6 Enzymatic Activities Gene Source Designation Organism EnzymeName Accession # EC Number Exemplary Use Fatty Acid Production IncreaseaccA E. coli, Acetyl-CoA AAC73296, 6.4.1.2 increase Malonyl-CoALactococci carboxylase, subunit A NP_414727 production(carboxyltransferase alpha) accB E. coli, Acetyl-CoA NP_417721 6.4.1.2increase Malonyl-CoA Lactococci carboxylase, subunit production B (BCCP:biotin carboxyl carrier protein) accC E. coli, Acetyl-CoA NP_4177226.4.1.2, increase Malonyl-CoA Lactococci carboxylase, subunit 6.3.4.14production C (biotin carboxylase) accD E. coli, Acetyl-CoA NP_4168196.4.1.2 increase Malonyl-CoA Lactococci carboxylase, subunit Dproduction (carboxyltransferase beta) fadD E. coli W3110 acyl-CoAsynthase AP_002424 2.3.1.86, increase Fatty acid 6.2.1.3 production fabAE. coli K12 β-hydroxydecanoyl NP_415474 4.2.1.60 increase fatty acyl-thioester ACP/CoA production dehydratase/isomerase fabB E. coli3-oxoacyl-[acyl- BAA16180 2.3.1.41 increase fatty acyl- carrier-protein]ACP/CoA production synthase I fabD E. coli K12 [acyl-carrier-protein]AAC74176 2.3.1.39 increase fatty acyl- S-malonyltransferase ACP/CoAproduction fabF E. coli K12 3-oxoacyl-[acyl- AAC74179 2.3.1.179 increasefatty acyl- carrier-protein] ACP/CoA production synthase II fabG E. coliK12 3-oxoacyl-[acyl- AAC74177 1.1.1.100 increase fatty acyl- carrierprotein] ACP/CoA production reductase fabH E. coli K12 3-oxoacyl-[acyl-AAC74175 2.3.1.180 increase fatty acyl- carrier-protein] ACP/CoAproduction synthase III fabI E. coli K12 enoyl-[acyl-carrier- NP_4158041.3.1.9 increase fatty acyl- protein] reductase ACP/CoA production fabRE. coli K12 Transcriptional NP_418398 none modulate unsaturatedRepressor fatty acid production fabV Vibrio choleraeenoyl-[acyl-carrier- YP_001217283 1.3.1.9 increase fatty acyl- protein]reductase ACP/CoA production fabZ E. coli K12 (3R)- NP_414722 4.2.1.-increase fatty acyl- hydroxymyristol ACP/CoA production acyl carrierprotein dehydratase fadE E. coli K13 acyl-CoA AAC73325 1.3.99.3, reducefatty acid dehydrogenase 1.3.99.- degradation fadD E. coli K12 acyl-CoAsynthetase NP_416319 6.2.1.3 reduce fatty acid degradation fadA E. coliK12 3-ketoacyl-CoA YP_02627 2.3.1.16 reduce fatty acid thiolasedegradation fadB E. coli K12 enoyl-CoA NP_418288 4.2.1.17, reduce fattyacid hydratase, 3-OH 5.1.2.3, degradation acyl-CoA epimerase/ 1.1.1.35dehydrogenase fadR E. coli transcriptional NP_415705 none Block orreverse fatty regulatory protein acid degradation Chain Length ControltesA (with or E. coli thioesterase - leader P0ADA1 3.1.2.-, C18 ChainLength without leader sequence is amino 3.1.1.5 sequence) acids 1-26tesA (without E. coli thioesterase AAC73596, 3.1.2.-, C18:1 Chain Lengthleader NP_415027 3.1.1.5 sequence) tesA (mutant E. coli thioesteraseL109P 3.1.2.-, <C18 Chain Length of E. coli 3.1.1.5 thioesterase Icomplexed with octanoic acid) fatB1 Umbellularia thioesterase Q416353.1.2.14 C12:0 Chain Length californica fatB2 Cuphea thioesteraseAAC49269 3.1.2.14 C8:0-C10:0 Chain hookeriana Length fatB3 Cupheathioesterase AAC72881 3.1.2.14 C14:0-C16:0 Chain hookeriana Length fatBCinnamomumcamphora thioesterase Q39473 3.1.2.14 C14:0 Chain Length fatBArabidopsis thioesterase CAA85388 3.1.2.14 C16:1 Chain Length thalianafatB1 Umbellularia thioesterase Q41635 3.1.2.14 C12:0 Chain Lengthcalifornica fatA1 Helianthus thioesterase AAL79361 3.1.2.14 C18:1 ChainLength annuus fatA Arabidopsis thioesterase NP_189147, 3.1.2.14 C18:1Chain Length thaliana NP_193041 fatA Brassica juncea thioesteraseCAC39106 3.1.2.14 C18:1 Chain Length fatA Cuphea thioesterase AAC728833.1.2.14 C18:1 Chain Length hookeriana tes Photbacterium thioesteraseYP_130990 3.1.2.14 Chain Length profundum tesB E. coli thioesteraseNP_414986 3.1.2.14 Chain Length fadM E. coli thioesterase NP_4149773.1.2.14 Chain Length yciA E. coli thioesterase NP_415769 3.1.2.14 ChainLength ybgC E. coli thioesterase NP_415264 3.1.2.14 Chain LengthSaturation Level Control Sfa E. coli Suppressor of fabA AAN79592, noneincrease mono- AAC44390 unsaturated fatty acids fabA E. coli K12β-hydroxydecanoyl NP_415474 4.2.1.60 produce unsaturated thioester fattyacids dehydratase/isomerase GnsA E. coli suppressors of the ABD18647.1none increase unsaturated secG null mutation fatty acid esters GnsB E.coli suppressors of the AAC74076.1 none increase unsaturated secG nullmutation fatty acid esters fabB E. coli 3-oxoacyl-[acyl- BAA161802.3.1.41 modulate unsaturated carrier-protein] fatty acid productionsynthase I des Bacillus subtilis D5 fatty acyl O34653 1.14.19 modulateunsaturated desaturase fatty acid production Ester Production AT3G51970Arabidopsis long-chain-alcohol NP_190765 2.3.1.26 ester productionthaliana O-fatty- acyltransferase ELO1 Pichia angusta Fatty acidelongase BAD98251 2.3.1.- produce very long chain length fatty acidsplsC Saccharomyces acyltransferase AAA16514 2.3.1.51 ester productioncerevisiae DAGAT/DGAT Arabidopsis diacylglycerol AAF19262 2.3.1.20 esterproduction thaliana acyltransferase hWS Homo sapiens acyl-CoA waxAAX48018 2.3.1.20 ester production alcohol acyltransferase aft1Acinetobacter sp. bifunctional wax AAO17391 2.3.1.20 ester productionADP1 ester synthase/acyl- CoA: diacylglycerol acyltransferase ES9Marinobacter wax ester synthase ABO21021 2.3.1.20 ester productionhydrocarbonoclasticus mWS Simmondsia wax ester synthase AAD38041 2.3.1.-ester production chinensis Fatty Alcohol Output thioesterases (seeincrease fatty acid/fatty above) alcohol production BmFAR Bombyxmori FAR(fatty alcohol BAC79425 1.1.1.- convert acyl-CoA to forming acyl-CoAfatty alcohol reductase) acr1 Acinetobacter sp. acyl-CoA reductaseYP_047869 1.2.1.42 reduce fatty acyl-CoA to ADP1 fatty aldehydes yqhD E.coli W3110 alcohol AP_003562 1.1.-.- reduce fatty aldehydes todehydrogenase fatty alcohols; increase fatty alcohol production alrAAcinetobacter sp. alcohol CAG70252 1.1.-.- reduce fatty aldehydes toADP1 dehydrogenase fatty alcohols BmFAR Bombyxmori FAR (fatty alcoholBAC79425 1.1.1.- reduce fatty acyl-CoA to forming acyl-CoA fatty alcoholreductase) GTNG_1865 Geobacillusthermo- Long-chain aldehyde YP_0011259701.2.1.3 reduce fatty aldehydes to denitrificans dehydrogenase fattyalcohols NG80-2 AAR Synechococcus Acyl-ACP reductase YP_400611 1.2.1.42reduce fatty acyl-ACP/ elongatus CoA to fatty aldehydes carBMycobacterium carboxylic acid YP_889972 6.2.1.3, reduce fatty acids tosmegmatis reductase protein 1.2.1.42 fatty aldehyde FadD E. coli K12acyl-CoA synthetase NP_416319 6.2.1.3 activates fatty acids to fattyacyl-CoAs atoB Erwiniacarotovora acetyl-CoA YP_049388 2.3.1.9 productionof butanol acetyltransferase hbd Butyrivibriofibrisolvens Beta- BAD514241.1.1.157 production of butanol hydroxybutyryl-CoA dehydrogenase CPE0095Clostridium crotonasebutyryl- BAB79801 4.2.1.55 production of butanolperfringens CoA dehydryogenase bcd Clostridium butyryl-CoA AAM145831.3.99.2 production of butanol beijerinckii dehydryogenase ALDHClostridium coenzyme A- AAT66436 1.2.1.3 production of butanolbeijerinckii acylating aldehyde dehydrogenase AdhE E. coli CFT073aldehyde-alcohol AAN80172 1.1.1.1 production of butanol dehydrogenase1.2.1.10 Fatty Alcohol Acetyl Ester Output thioesterases (see modifyoutput above) acr1 Acinetobacter sp. acyl-CoA reductase YP_0478691.2.1.42 modify output ADP1 yqhD E. Coli K12 alcohol AP_003562 1.1.-.-modify output dehydrogenase AAT Fragaria x alcohol O- AAG13130 2.3.1.84modify output ananassa acetyltransferase Terminal Olefin Output OleTJeotgalicoccus Fatty acid HQ709266 1.11.2.4 decarboxylate fatty acidssp. decarboxylase Product Export AtMRP5 Arabidopsis Arabidopsis thalianaNP_171908 none modify product export thaliana multidrug resistance-amount associated AmiS2 Rhodococcus sp. ABC transporter JC5491 nonemodify product export AmiS2 amount AtPGP1 Arabidopsis Arabidopsisthaliana NP_181228 none modify product export thaliana p glycoprotein 1amount AcrA CandidatusPro- putative multidrug- CAF23274 none modifyproduct export tochlamydiaamoebophila efflux transport amount UWE25protein acrA AcrB CandidatusProto- probable multidrug- CAF23275 nonemodify product export chlamydiaamoebophila UWE25 efflux transport amountprotein, acrB TolC Francisellatularensis subsp. Outer membrane ABD59001none modify product export novicida protein [Cell amount envelopebiogenesis, AcrE Shigellasonnei transmembrane YP_312213 none modifyproduct export Ss046 protein affects amount septum formation and cellmembrane permeability AcrF E. coli Acriflavine P24181 none modifyproduct export resistance protein F amount tll1619 Thermosynechococcusmultidrug efflux NP_682409.1 none modify product export elongatus [BP-1]transporter amount tll0139 Thermosynechococcus multidrug effluxNP_680930.1 none modify product export elongatus [BP-1] transporteramount Fermentation replication increase output checkpoint efficiencygenes umuD Shigellasonnei DNA polymerase V, YP_310132 3.4.21.- increaseoutput Ss046 subunit efficiency umuC E. coli DNA polymerase V, ABC422612.7.7.7 increase output subunit efficiency pntA, pntB ShigellaflexneriNADH: NADPH P07001, 1.6.1.2 increase output transhydrogenase P0AB70efficiency (alpha and beta subunits) Other fabK Streptococcustrans-2-enoyl-ACP AAF98273 1.3.1.9 Contributes to fatty acid pneumoniaereductase II biosynthesis fabL Bacillus enoyl-(acyl carrier AAU398211.3.1.9 Contributes to fatty acid licheniformis protein) reductasebiosynthesis DSM 13 fabM Streptococcus trans-2, cis-3- DAA05501 4.2.1.17Contributes to fatty acid mutans decenoyl-ACP biosynthesis isomerase

TABLE 7 Examples of Amino Transferases/Transaminases (EC 2.6.1) andAmine Dehydrogenases (EC 1.4.9, EC 1.4.98, EC 1.4.99) Designation/NameFunction Organism Accession # Beta alanine-pyruvate Beta-alanine:pyruvate Pseudomonas aeruginosa PA7 YP_001345604 transaminasetransaminase ygjG Putrescine aminotransferase Escherichia coli MG1655NP_417544 gabT 5-aminovalerate transaminase Pseudomonas aeruginosa PA01AAG03655 Lat L-lysine 6-transaminase Mycobacterium tuberculosisNP_217807 H37Rv GABA-T 4-aminobutyrate transaminase Sus scrofa NP_999428Ald Alanine dehydrogenase Bacillus subtilis subsp. BAI86717 nattoBEST195 gdhA Glutamate dehydrogenase Escherichia coli MG1655 NP_416275(NADPH) Gdh Glutamate dehydrogenase Peptoniphilus asaccharolyticusAAA25611 (NADH) L-lysine L-lysine 6-dehydrogenase Achromobacterdenitrificans AAZ94428 6-dehydrogenase mauRFBEDACJGMN Methylaminedehydrogenase Paracoccus denitrificans P52685.1 P29897.2 P29894.1P29896.2 P29895.2 P22619.2 P22364.1 P22566.2 ABL72797.1 ABL72798.1AAA86469.1

Microbial Host Cells and their Cultures

The microorganisms of the disclosure function as microbial host cellsand encompass one or more polynucleotide sequences that include an openreading frame encoding at least one exogenous biosynthetic enzyme of thepresent disclosure. In one embodiment, a fatty amine composition isproduced by culturing host cells that express an exogenous biosyntheticenzyme (e.g., aminotransferases/transaminases or amine dehydrogenases oramine oxidases) in the presence of a carbon source under conditionseffective to express the fatty amines. In another embodiment, a fattyamine composition is produced by culturing host cells that express oneor more of an exogenous biosynthetic enzyme (e.g.,aminotransferases/transaminases or amine dehydrogenases or amineoxidases in combination with one or more aldehyde generating enzymessuch as a CAR (e.g., CarB) and/or a TE (e.g., TesA, 'tesA) in thepresence of a carbon source under conditions effective to express thefatty amines. In another embodiment, a fatty amine composition isproduced by culturing host cells that express one or more of anexogenous biosynthetic enzyme (e.g., aminotransferases/transaminases oramine dehydrogenases or amine oxidases in combination with one or morealdehyde generating enzymes such as an acyl-ACP reductase (AAR) (e.g.,from Synechococcus elongatus PCC7942) in the presence of a carbon sourceunder conditions effective to express the fatty amines. In anotherembodiment, a fatty amine composition is produced by culturing hostcells that express one or more of an exogenous biosynthetic enzyme(e.g., aminotransferases/transaminases or amine dehydrogenases or amineoxidases in combination with one or more aldehyde generating enzymessuch as an acyl-CoA reductase (e.g., Acr1) in the presence of a carbonsource under conditions effective to express the fatty amines. Inanother embodiment, a fatty amine composition is produced by culturinghost cells that express one or more of an exogenous biosynthetic enzyme(e.g., aminotransferases/transaminases or amine dehydrogenases or amineoxidases in combination with one or more aldehyde generating enzymessuch as a phosphopanthetheinyl transferase (PPTase) in the presence of acarbon source under conditions effective to express the fatty amines.

Expression of the biosynthetic enzymes results in production of fattyamines with increased yields of fatty amines and/or fatty aminecompositions or blends thereof. In one embodiment, expression of anaminotransferase or amine dehydrogenase polypeptide in the host cellresults in a high yield of fatty amines or compositions thereof. Inanother embodiment, expression of an aminotransferase or aminedehydrogenase polypeptide in combination with one or more aldehydegenerating enzymes in the host cell results in a high yield of fattyamines and compositions thereof. In another embodiment, expression of anamine oxidase in combination with one or more aldehyde generatingenzymes in the host cell results in high yields of fatty amines andcompositions thereof. In some embodiments, the biosynthetic enzymes areexogenously expressed in the cell.

The host cells or microorganisms of the disclosure may include hoststrains or host cells that are further genetically engineered to containalterations in order to test the efficiency of specific mutations ormanipulations on enzymatic activities (i.e., recombinant cells ormicroorganisms). Various optional genetic manipulations and alterationscan be used interchangeably from one host cell to another, depending onwhat native enzymatic pathways are present in the original host cell. Inone embodiment, a host strain can be used for testing the expression ofan aminotransferase or amine dehydrogenase polypeptide in combinationwith an aldehyde generating polypeptide. A host strain may encompasses anumber of genetic alterations in order to test specific variables,including but not limited to, culture conditions including fermentationcomponents, carbon source (e.g., feedstock), temperature, pressure,reduced culture contamination conditions, and oxygen levels.

In one embodiment, a host strain encompasses an optional fadE and fhuAdeletion. Acyl-CoA dehydrogenase (FadE) is an enzyme that is importantfor metabolizing fatty acids. It catalyzes the second step in fatty acidutilization (beta-oxidation), which is the process of breaking longchains of fatty acids (acyl-CoAs) into acetyl-CoA molecules. Morespecifically, the second step of the β-oxidation cycle of fatty aciddegradation in bacteria is the oxidation of acyl-CoA to 2-enoyl-CoA,which is catalyzed by FadE. When E. coli lacks FadE, it cannot grow onfatty acids as a carbon source but it can grow on acetate. The inabilityto utilize fatty acids of any chain length is consistent with thereported phenotype of fadE strains, i.e., fadE mutant strains where FadEfunction is disrupted. The fadE gene can be optionally knocked out orattenuated to assure that acyl-CoAs, which may be intermediates in afatty amine pathway, can accumulate in the cell such that all acyl-CoAscan be efficiently converted to fatty amines. However, fadE attenuationis optional when sugar is used as a carbon source since under suchcondition expression of FadE is likely repressed and FadE therefore mayonly be present in small amounts and not able to efficiently competewith ester synthase for acyl-CoA substrates. FadE is repressed due tocatabolite repression. E. coli and other microbes prefer to consumesugar over fatty acids, so when both sources are available sugar isconsumed first by repressing the fad regulon (see D. Clark, J Bacteriol.(1981) 148(2):521-6)). Moreover, the absence of sugars induces FadEexpression. Acyl-CoA intermediates could be lost to the beta oxidationpathway since the proteins expressed by the fad regulon (including FadE)are up-regulated and will efficiently compete for acyl-CoAs. Thus, itcan be beneficial under certain circumstances to have the fadE geneknocked out or attenuated. Since many carbon sources are carbohydratebased, it is optional to attenuate FadE. The gene fhuA codes for theTonA protein, which is an energy-coupled transporter and receptor in theouter membrane of E. coli (V. Braun (2009) J Bacteriol.191(11):3431-3436). Its deletion is optional. The fhuA deletion allowsthe cell to become more resistant to phage attack which can bebeneficial in certain fermentation conditions. Thus, it may be desirableto delete fhuA in a host cell that is likely subject to potentialcontamination during fermentation runs.

In another embodiment, the host strain (supra) may also encompassoptional overexpression of one or more of the following genes includingfadR, fabA, fabD, fabG, fabH, fabV, and/or fabF. Examples of such genesare fadR from Escherichia coli, fabA from Salmonella typhimurium(NP_460041), fabD from Salmonella typhimurium (NP_460164), fabG fromSalmonella typhimurium (NP_460165), fabH from Salmonella typhimurium(NP_460163), fabV from Vibrio cholera (YP_001217283), and fabF fromClostridium acetobutylicum (NP_350156). The optional overexpression ofone or more of these genes, which code for enzymes and regulators infatty acid biosynthesis, can serve to increase the titer of fatty-acidderivative compounds under various culture conditions.

In another embodiment, E. coli strains are used as host cells for theproduction of fatty amines. Similarly, these host cells may provideoptional overexpression of one or more biosynthesis genes (i.e., genescoding for enzymes and regulators of fatty acid biosynthesis) that canincrease the titer of fatty-acid derivative compounds such as fattyamines under various culture conditions including, but not limited to,fadR, fabA, fabD, fabG, fabH, fabV and/or fabF. Examples of geneticalterations include fadR from Escherichia coli, fabA from Salmonellatyphimurium (NP_460041), fabD from Salmonella typhimurium (NP_460164),fabG from Salmonella typhimurium (NP_460165), fabH from Salmonellatyphimurium (NP_460163), fabV from Vibrio cholera (YP_001217283), andfabF from Clostridium acetobutylicum (NP_350156).

In some embodiments, the host cells or microorganisms that are used toexpress the biosynthetic enzymes (e.g., aminotransferases or aminedehydrogenases in combination with aldehyde generating enzymes such asCAR and/or TE and/or AAR and/or PPTase) may further express genes thatencompass certain enzymatic activities that can increase the productionto one or more particular fatty acid derivative(s) such as fatty esters,fatty alcohols, fatty amines, fatty aldehydes, bifunctional fatty acidderivatives, diacids and the like. In one embodiment, the host cell hasthioesterase activity (E.C. 3.1.2.* or E.C. 3.1. 2.14 or E.C. 3.1.1.5)for the production of fatty acids which can be increased byoverexpressing the gene. In another embodiment, the host cell has estersynthase activity (E.C. 2.3.1.75) for the production of fatty esters. Inanother embodiment, the host cell has acyl-ACP reductase (AAR) (E.C.1.2.1.80) activity and/or alcohol dehydrogenase activity (E.C. 1.1.1.1.)and/or fatty alcohol acyl-CoA reductase (FAR) (E.C. 1.1.1.*) activityand/or carboxylic acid reductase (CAR) (EC 1.2.99.6) activity for theproduction of fatty alcohols. In another embodiment, the host cell hasacyl-ACP reductase (AAR) (E.C. 1.2.1.80) activity for the production offatty aldehydes. In another embodiment, the host cell has acyl-ACPreductase (AAR) (E.C. 1.2.1.80) activity and decarbonylase activity forthe production of alkanes and alkenes. In another embodiment, the hostcell has acyl-CoA reductase (E.C. 1.2.1.50) activity, acyl-CoA synthase(FadD) (E.C. 2.3.1.86) activity, and thioesterase (E.C. 3.1.2.* or E.C.3.1. 2.14 or E.C. 3.1.1.5) activity for the production of fattyalcohols. In another embodiment, the host cell has ester synthaseactivity (E.C. 2.3.1.75), acyl-CoA synthase (FadD) (E.C. 2.3.1.86)activity, and thioesterase (E.C. 3.1.2.* or E.C. 3.1. 2.14 or E.C.3.1.1.5) activity for the production of fatty esters. In anotherembodiment, the host cell has OleA activity for the production ofketones. In another embodiment, the host cell has OleBCD activity forthe production of internal olefins. In another embodiment, the host cellhas acyl-ACP reductase (AAR) (E.C. 1.2.1.80) activity and alcoholdehydrogenase activity (E.C. 1.1.1.1.) for the production of fattyalcohols. In another embodiment, the host cell has thioesterase (E.C.3.1.2.* or E.C. 3.1. 2.14 or E.C. 3.1.1.5) activity and decarboxylaseactivity for making terminal olefins. The expression of enzymaticactivities in microorganisms and microbial cells is taught by U.S. Pat.Nos. 8,097,439; 8,110,093; 8,110,670; 8,183,028; 8,268,599; 8,283,143;8,232,924; 8,372,610; and 8,530,221, which are incorporated herein byreference.

In other embodiments, the host cells or microorganisms that are used toexpress the biosynthetic enzymes (e.g., aminotransferases or aminedehydrogenases in combination with aldehyde generating enzymes such asCAR and/or TE and/or AAR and/or PPTase) will include certain nativeenzyme activities that are upregulated or overexpressed in order toproduce one or more particular fatty acid derivative(s) such as fattyamines. In one embodiment, the host cell has a native thioesterase (E.C.3.1.2.* or E.C. 3.1. 2.14 or E.C. 3.1.1.5) activity for the productionof fatty acids which can be increased by overexpressing the thioesterasegene.

The present disclosure includes host strains or microorganisms thatexpress genes that code for the biosynthetic enzymes (e.g.,aminotransferases or amine dehydrogenases in combination with aldehydegenerating enzymes such as CAR and/or TES and/or AAR and/or PPTase). Inone embodiment, at least one biosynthetic enzyme is exogenouslyexpressed in the host cell. For example, the host cell may express anexogenous aminotransferase in order to produce fatty amines. In anotherembodiment, one or more biosynthetic enzymes are exogenously expressedin the host cell. For example, the host cell may express an exogenousaminotransferase and an exogenous carboxylic acid reductase (CAR) inorder to produce fatty amines. In still another embodiment, one or morebiosynthetic enzymes are exogenously expressed in the host cell incombination with one or more biosynthetic enzymes that are overexpressedin the host cell. For example, the host cell may express an exogenousaminotransferase and an exogenous carboxylic acid reductase (CAR) incombination with an exogenous and/or overexpressed thioesterase in orderto produce fatty amines. The thioesterase may be an exogenouslyexpressed thioesterase. Alternatively, the thioesterase may be a nativethioesterase that is overexpressed or transcriptionally upregulated inthe cell via a particularly strong promoter or other molecular biologytechniques that are well known to those of skill in the art. Therecombinant host cells produce fatty amines and compositions and blendsthereof. The fatty amines are typically recovered from the culturemedium and/or are isolated from the host cells. In one embodiment, thefatty amines are recovered from the culture medium (extracellular). Inanother embodiment, the fatty amines are isolated from the host cells(intracellular). In another embodiment, the fatty amines are recoveredfrom the culture medium and isolated from the host cells. The fattyamine composition produced by a host cell can be analyzed using methodsknown in the art, for example, GC-FID, in order to determine thedistribution of particular fatty amines as well as chain lengths anddegree of saturation of the components of the fatty amine composition.

Examples of host cells that function as microorganisms (e.g., microbialcells), include but are not limited to cells from the genus Escherichia,Bacillus, Lactobacillus, Zymomonas, Rhodococcus, Pseudomonas,Aspergillus, Trichoderma, Neurospora, Fusarium, Humicola, Rhizomucor,Kluyveromyces, Pichia, Mucor, Myceliophtora, Penicillium, Phanerochaete,Pleurotus, Trametes, Chrysosporium, Saccharomyces, Stenotrophamonas,Schizosaccharomyces, Yarrowia, or Streptomyces. In some embodiments, thehost cell is a Gram-positive bacterial cell. In other embodiments, thehost cell is a Gram-negative bacterial cell. In some embodiments, thehost cell is an E. coli cell. In some embodiment, the host cell is an E.coli B cell, an E. coli C cell, an E. coli K cell, or an E. coli W cell.In other embodiments, the host cell is a Bacillus lentus cell, aBacillus brevis cell, a Bacillus stearothermophilus cell, a Bacilluslichenoformis cell, a Bacillus alkalophilus cell, a Bacillus coagulanscell, a Bacillus circulans cell, a Bacillus pumilis cell, a Bacillusthuringiensis cell, a Bacillus clausii cell, a Bacillus megaterium cell,a Bacillus subtilis cell, or a Bacillus amyloliquefaciens cell.

In still other embodiments, the host cell is a Trichoderma koningiicell, a Trichoderma viride cell, a Trichoderma reesei cell, aTrichoderma longibrachiatum cell, an Aspergillus awamori cell, anAspergillus fumigates cell, an Aspergillus foetidus cell, an Aspergillusnidulans cell, an Aspergillus niger cell, an Aspergillus oryzae cell, aHumicola insolens cell, a Humicola lanuginose cell, a Rhodococcus opacuscell, a Rhizomucor miehei cell, or a Mucor michei cell. In yet otherembodiments, the host cell is a Streptomyces lividans cell or aStreptomyces murinus cell. In yet other embodiments, the host cell is anActinomycetes cell. In some embodiments, the host cell is aSaccharomyces cerevisiae cell.

In other embodiments, the host cell is a cell from a eukaryotic plant,algae, cyanobacterium, green-sulfur bacterium, green non-sulfurbacterium, purple sulfur bacterium, purple non-sulfur bacterium,extremophile, yeast, fungus, an engineered organism thereof, or asynthetic organism. In some embodiments, the host cell islight-dependent or fixes carbon. In some embodiments, the host cell hasautotrophic activity.

In some embodiments, the host cell has photoautotrophic activity, suchas in the presence of light. In some embodiments, the host cell isheterotrophic or mixotrophic in the absence of light. In certainembodiments, the host cell is a cell from Arabidopsis thaliana, Panicumvirgatum, Miscanthus giganteus, Zea mays, Botryococcuse braunii,Chlamydomonas reinhardtii, Dunaliela salina, Synechococcus Sp. PCC 7002,Synechococcus Sp. PCC 7942, Synechocystis Sp. PCC 6803,Thermosynechococcus elongates BP-1, Chlorobium tepidum, Chlorojlexusauranticus, Chromatiumm vinosum, Rhodospirillum rubrum, Rhodobactercapsulatus, Rhodopseudomonas palusris, Clostridium ljungdahlii,Clostridium thermocellum, Penicillium chrysogenum, Pichia pastoris,Saccharomyces cerevisiae, Schizosaccharomyces pombe, Pseudomonasfluorescens, or Zymomonas mobilis.

In one particular embodiment, the microbial cell is from a cyanobacteriaincluding, but not limited to, Prochlorococcus, Synechococcus,Synechocystis, Cyanothece, and Nostoc punctiforme. In anotherembodiment, the microbial cell is from a specific cyanobacterial speciesincluding, but not limited to, Synechococcus elongatus PCC7942,Synechocystis sp. PCC6803, and Synechococcus sp. PCC7001.

Methods of Making Recombinant Host Cells and Cultures

Various methods well known in the art can be used to engineer host cellsto produce fatty amines and/or fatty amine compositions or blends. Themethods can include the use of vectors, preferably expression vectors,including a nucleic acid encoding the biosynthetic enzyme (e.g.,aminotransferases or amine dehydrogenases alone or in combination withaldehyde generating enzymes such as CAR and/or TE and/or AAR and/orPPTase), as described herein. Those skilled in the art will appreciate avariety of viral and non-viral vectors can be used in the methodsdescribed herein.

In some embodiments of the present disclosure, a higher titer of fattyamines in a particular composition is a higher titer of a particulartype of fatty amine or a combination of fatty amines produced by arecombinant host cell culture relative to the titer of the same fattyacid amine or combination of fatty amine produced by a control cultureof a corresponding wild-type host cell. In some embodiments,biosynthetic polypeptides (e.g., aminotransferases or aminedehydrogenases alone or in combination with polypeptides of aldehydegenerating enzymes such as CAR and/or TE and/or AAR and/or PPTase) areprovided to the host cell by way of a recombinant vector, which mayinclude a promoter operably linked to a specific polynucleotide sequencethat codes for a specific biosynthetic polypeptide. In certainembodiments, the promoter is a developmentally-regulated, anorganelle-specific, a tissue-specific, an inducible, a constitutive, ora cell-specific promoter. The recombinant vector typically comprises atleast one sequence selected from an expression control sequenceoperatively coupled to the polynucleotide sequence; a selection markeroperatively coupled to the polynucleotide sequence; a marker sequenceoperatively coupled to the polynucleotide sequence; a purificationmoiety operatively coupled to the polynucleotide sequence; a secretionsequence operatively coupled to the polynucleotide sequence; and atargeting sequence operatively coupled to the polynucleotide sequence.The polynucleotide sequences, comprising open reading frames encodingproteins and operably-linked regulatory sequences can be integrated intoa chromosome of the recombinant host cells, incorporated in one or moreplasmid expression system resident in the recombinant host cells, orboth.

The expression vectors include a polynucleotide sequence as describedherein in a form suitable for expression of the polynucleotide sequencein a host cell. It will be appreciated by those skilled in the art thatthe design of the expression vector can depend on such factors as thechoice of the host cell to be transformed, the level of expression ofpolypeptide desired, etc. The expression vectors described herein can beintroduced into host cells to produce polypeptides, including fusionpolypeptides, encoded by the polynucleotide sequences as describedherein. Expression of genes encoding polypeptides in prokaryotes, forexample, E. coli, is most often carried out with vectors containingconstitutive or inducible promoters directing the expression of eitherfusion or non-fusion polypeptides. Suitable expression systems for bothprokaryotic and eukaryotic cells are well known in the art (see, e.g.,Sambrook et al., “Molecular Cloning: A Laboratory Manual,” secondedition, Cold Spring Harbor Laboratory, (1989)). In certain embodiments,a polynucleotide sequence of the disclosure is operably linked to apromoter derived from bacteriophage T5. In one embodiment, the host cellis a yeast cell. In this embodiment, the expression vector is a yeastexpression vector. Vectors can be introduced into prokaryotic oreukaryotic cells via a variety of art-recognized techniques forintroducing foreign nucleic acid (e.g., DNA) into a host cell. Suitablemethods for transforming or transfecting host cells can be found in, forexample, Sambrook et al. (supra).

For stable transformation of bacterial cells, it is known that,depending upon the expression vector and transformation technique used,only a small fraction of cells will take-up and replicate the expressionvector. In order to identify and select these transformants, a gene thatencodes a selectable marker (e.g., resistance to an antibiotic) can beintroduced into the host cells along with the gene of interest.Selectable markers include those that confer resistance to drugs suchas, but not limited to, ampicillin, kanamycin, chloramphenicol, ortetracycline. Nucleic acids encoding a selectable marker can beintroduced into a host cell on the same vector as that encoding apolypeptide described herein or can be introduced on a separate vector.Cells stably transformed with the introduced nucleic acid can beidentified by growth in the presence of an appropriate selection drug.

Culture and Fermentation of Recombinant Host Cells

As used herein, the term “fermentation” broadly refers to the conversionof organic materials into target substances by host cells, for example,the conversion of a carbon source by recombinant host cells into fattyamines or derivatives thereof by propagating a culture of therecombinant host cells in a media comprising the carbon source. As usedherein, the term “conditions permissive for the production” means anyconditions that allow a host cell to produce a desired product, such asa fatty amine or fatty amine composition or blend. Similarly, the term“conditions in which the polynucleotide sequence of a vector isexpressed” means any conditions that allow a host cell to synthesize apolypeptide. Suitable conditions include, for example, fermentationconditions. Fermentation conditions can comprise many parameters,including but not limited to temperature ranges, levels of aeration,feed rates and media composition. Each of these conditions, individuallyand in combination, allows the host cell to grow. Fermentation can beaerobic, anaerobic, or variations thereof (such as micro-aerobic).Exemplary culture media include broths or gels. Generally, the mediumincludes a carbon source that can be metabolized by a host celldirectly. In addition, enzymes can be used in the medium to facilitatethe mobilization (e.g., the depolymerization of starch or cellulose tofermentable sugars) and subsequent metabolism of the carbon source.

For small scale production, the engineered host cells can be grown inbatches of, for example, about 100 μL, 200 μL, 300 μL, 400 μL, 500 μL, 1mL, 5 mL, 10 mL, 15 mL, 25 mL, 50 mL, 75 mL, 100 mL, 500 mL, 1 L, 2 L, 5L, or 10 L; fermented; and induced to express a desired polynucleotidesequence, such as a polynucleotide sequence encoding anaminotransferase/transaminase or an amine dehydrogenase or an amineoxidase polypeptide alone or in combination with an aldehyde-generatingpolynucleotide sequence encoding a CAR and/or a TE and/or an AAR and/ora PPtase polypeptide. For large scale production, the engineered hostcells can be grown in cultures having volume batches of about 10 L, 100L, 1000 L, 10,000 L, 100,000 L, 1,000,000 L or larger; fermented; andinduced to express a desired polynucleotide sequence. In one preferredembodiment, the fatty amines and fatty amine derivative compositionsdescribed herein are found in the extracellular environment of therecombinant host cell culture and can be readily isolated from theculture medium. In another embodiment, the fatty amines and fatty aminederivative compositions described herein are found in the intracellularenvironment of the recombinant host cells grown in culture. A fattyamine or derivative thereof may be secreted by the recombinant hostcell, transported into the extracellular environment or passivelytransferred into the extracellular environment of the recombinant hostcell culture. The fatty amine composition may be isolated from arecombinant host cell culture using routine methods known in the art.

Screening Recombinant Host Cells

In one embodiment of the present disclosure, the activity of anaminotransferase/transaminase or an amine dehydrogenase or an amineoxidase polypeptide is determined by culturing recombinant host cellsencompassing one or more aminotransferase/transaminase or aminedehydrogenase or amine oxidase polypeptide sequences (optionally incombination with one or more aldehyde-generating polypeptides), followedby screening to identify characteristics of, for example, fatty aminecompositions produced by the recombinant host cells; for example, titer,yield and productivity of fatty amines and compositions and blendsthereof. In another embodiment, the activity of aaminotransferase/transaminase or amine dehydrogenase or amine oxidasepolypeptide is determined by culturing recombinant host cellsencompassing one or more aminotransferase/transaminase or aminedehydrogenase or amine oxidase polynucleotide sequences, followed byscreening to identify characteristics of, for example, fatty aminecompositions produced by the recombinant host cells; for example: titer,yield and productivity of fatty amines and compositions and blendsthereof. The aminotransferase/transaminase or amine dehydrogenase oramine oxidase polypeptides and fragments thereof can be assayed fortheir activity in a cell and/or improved/increased production ofamine-derived compounds using routine methods known in the art. Forexample, an aminotransferase/transaminase or an amine dehydrogenase oran amine oxidase polypeptide or fragment thereof is contacted with asubstrate in vivo (e.g., a fatty aldehyde produced by coexpressing CARand/or TE and/or AAR and/or PPTase in the cell) under conditions thatallow the polypeptide to function and carry out its enzymatic activity.A decrease in the level of the substrate or an increase in the level ofa fatty amine or a fatty amine composition can be measured to determinethe activity of the aminotransferase/transaminase or amine dehydrogenaseor amine oxidase. Alternatively, a cell expressing anaminotransferase/transaminase or an amine dehydrogenase or an amineoxidase polypeptide or fragment thereof can be fed with a fatty aldehydesubstrate under conditions that still allow the polypeptide to functionand carry out its enzymatic activity. An increase in the level of afatty amine or a fatty amine composition can then be measured todetermine the activity of the aminotransferase/transaminase or aminedehydrogenase or amine oxidase.

Products Derived from Recombinant Host Cells

As used herein, “fraction of modem carbon” or fM has the same meaning asdefined by National Institute of Standards and Technology (NIST)Standard Reference Materials (SRMs 4990B and 4990C, known as oxalicacids standards HOxI and HOxII, respectively. The fundamental definitionrelates to 0.95 times the 14C/12C isotope ratio HOxI (referenced to AD1950). This is roughly equivalent to decay-corrected pre-IndustrialRevolution wood. For the current living biosphere (plant material), fMis approximately 1.1.

Bioproducts (e.g., the fatty amine compositions produced in accordancewith the present disclosure) comprising biologically produced organiccompounds, and in particular, the fatty amine compositions producedusing the biosynthetic pathway herein, have been produced from renewablesources and, as such, are new compositions of matter. These newbioproducts can be distinguished from organic compounds derived frompetrochemical carbon on the basis of dual carbon-isotopic fingerprintingor ¹⁴C dating. Additionally, the specific source of biosourced carbon(e.g., glucose vs. glycerol, etc.) can be determined by dualcarbon-isotopic fingerprinting (see, e.g., U.S. Pat. No. 7,169,588). Theability to distinguish bioproducts from petroleum based organiccompounds is beneficial in tracking these materials in commerce. Forexample, organic compounds or chemicals comprising both biologicallybased and petroleum based carbon isotope profiles may be distinguishedfrom organic compounds and chemicals made only of petroleum basedmaterials. Hence, the bioproducts produced herein can be followed ortracked in commerce on the basis of their unique carbon isotope profile.Bioproducts can be distinguished from petroleum based organic compoundsby comparing the stable carbon isotope ratio (¹³C/¹²C) in each sample.The ¹³C/¹²C ratio in a given bioproduct is a consequence of the ¹³C/¹²Cratio in atmospheric carbon dioxide at the time the carbon dioxide isfixed. It also reflects the precise metabolic pathway. Regionalvariations also occur. Petroleum, C3 plants (the broadleaf), C4 plants(the grasses), and marine carbonates all show significant differences in¹³C/¹²C and the corresponding δ¹³C values. Both C4 and C3 plants exhibita range of ¹³C/¹²C isotopic ratios, but typical values are about −7 toabout −13 per mil for C4 plants and about −19 to about −27 per mil forC3 plants (see, e.g., Stuiver et al. (1977) Radiocarbon 19:355). Coaland petroleum fall generally in this latter range.δ¹3C(%_(o))=[(¹³C/¹²C)sample−(¹³C/¹²C)standard]/(¹³C/¹²C)standard×1000

A series of alternative RMs have been developed in cooperation with theIAEA, USGS, NIST, and other selected international isotope laboratories.Notations for the per mil deviations from PDB is δ¹³C. Measurements aremade on CO₂ by high precision stable ratio mass spectrometry (IRMS) onmolecular ions of masses 44, 45, and 46. The compositions describedherein include fatty amine compositions and products produced by any ofthe methods described herein. Specifically, fatty amine composition orproduct can have a δ¹³C of about −28 or greater, about −27 or greater,−20 or greater, −18 or greater, −15 or greater, −13 or greater, −10 orgreater, or −8 or greater. For example, the fatty amine composition orproduct can have a δ¹³C of about −30 to about −15, about −27 to about−19, about −25 to about −21, about −15 to about −5, about −13 to about−7, or about −13 to about −10. In other instances, the fatty aminecomposition or product can have a δ¹³C of about −10, −11, −12, or −12.3.Fatty amine compositions and products produced in accordance with thedisclosure herein can also be distinguished from petroleum based organiccompounds by comparing the amount of ¹⁴C in each compound. Because ¹⁴Chas a nuclear half-life of 5730 years, petroleum based fuels containing“older” carbon can be distinguished from fatty amine compositions andbioproducts which contain “newer” carbon (see, e.g., Currie, “SourceApportionment of Atmospheric Particles”, Characterization ofEnvironmental Particles, J. Buffle and H. P. van Leeuwen, Eds., 1 ofVol. I of the IUPAC Environmental Analytical Chemistry Series (LewisPublishers, Inc.) 3-74, (1992)).

The basic assumption in radiocarbon dating is that the constancy of ¹⁴Cconcentration in the atmosphere leads to the constancy of ¹⁴C in livingorganisms. However, because of atmospheric nuclear testing since 1950and the burning of fossil fuel since 1850, ¹⁴C has acquired a second,geochemical time characteristic. Its concentration in atmospheric CO₂,and hence in the living biosphere, approximately doubled at the peak ofnuclear testing, in the mid-1960s. It has since been gradually returningto the steady-state cosmogenic (atmospheric) baseline isotope rate(¹⁴C/¹²C) of about 1.2×10-12, with an approximate relaxation “half-life”of 7-10 years. This latter half-life must not be taken literally;rather, one must use the detailed atmospheric nuclear input/decayfunction to trace the variation of atmospheric and biospheric ¹⁴C sincethe onset of the nuclear age. It is this latter biospheric ¹⁴C timecharacteristic that holds out the promise of annual dating of recentbiospheric carbon. ¹⁴C can be measured by accelerator mass spectrometry(AMS), with results given in units of “fraction of modern carbon” (fM).The fatty amine compositions and products described herein includebioproducts that can have an fM ¹⁴C of at least about 1. For example,the bioproduct of the disclosure can have an fM ¹⁴C of at least about1.01, an fM ¹⁴C of about 1 to about 1.5, an fM ¹⁴C of about 1.04 toabout 1.18, or an fM ¹⁴C of about 1.111 to about 1.124.

Another measurement of ¹⁴C is known as the percent of modern carbon(pMC). For an archaeologist or geologist using ¹⁴C dates, AD 1950 equals“zero years old”. This also represents 100 pMC. “Bomb carbon” in theatmosphere reached almost twice the normal level in 1963 at the peak ofthermo-nuclear weapons. Its distribution within the atmosphere has beenapproximated since its appearance, showing values that are greater than100 pMC for plants and animals living since AD 1950. It has graduallydecreased over time with today's value being near 107.5 pMC. This meansthat a fresh biomass material, such as corn, would give a ¹⁴C signaturenear 107.5 pMC. Petroleum based compounds will have a pMC value of zero.Combining fossil carbon with present day carbon will result in adilution of the present day pMC content. By presuming 107.5 pMCrepresents the ¹⁴C content of present day biomass materials and 0 pMCrepresents the ¹⁴C content of petroleum based products, the measured pMCvalue for that material will reflect the proportions of the twocomponent types. For example, a material derived 100% from present daysoybeans would give a radiocarbon signature near 107.5 pMC. If thatmaterial was diluted 50% with petroleum based products, it would give aradiocarbon signature of approximately 54 pMC. A biologically basedcarbon content is derived by assigning “100%” equal to 107.5 pMC and“0%” equal to 0 pMC. For example, a sample measuring 99 pMC will give anequivalent biologically based carbon content of 93%. This value isreferred to as the mean biologically based carbon result and assumes allthe components within the analyzed material originated either frompresent day biological material or petroleum based material. Abioproduct comprising one or more fatty amines as described herein canhave a pMC of at least about 50, 60, 70, 75, 80, 85, 90, 95, 96, 97, 98,99, or 100. In other instances, a fatty ester composition describedherein can have a pMC of between about 50 and about 100; about 60 andabout 100; about 70 and about 100; about 80 and about 100; about 85 andabout 100; about 87 and about 98; or about 90 and about 95. In yet otherinstances, a fatty amine composition described herein can have a pMC ofabout 90, 91, 92, 93, 94, or 94.2.

Fatty Amine Compositions

The structure of fatty amines is based on one or more C8 to C24aliphatic alkyl groups (R=C₈-C₂₄) and one or more amine (N) orquaternary ammonium. The aliphatic alkyl chain is strongly hydrophobicwhile the amine is hydrophilic. Thus, the fatty amine has an amphiphilicnature as a molecule (containing both hydrophobic and hydrophilicentities). When dissolved in water or other solvents, fatty amines formmicelles because one part of the molecule is repelled by the solvent. Assuch, fatty amines are cationic surface-active compounds (i.e.,surfactants that are characterized by their hydrophilic moiety) whichstrongly adhere to surfaces by either physical or chemical bonding, thusmodifying surface properties. The surface active properties of fattyamines are emulsification, wetting, foaming, and thickening. Inaddition, fatty amines have adsorptive properties including softening,adhesion, lubrication, corrosion inhibition, anti-static properties andhydrophobation; as well as reactive properties including ion exchange,decolorization, and flocculation.

The present disclosure contemplates the production of fatty amines andderivatives thereof that are useful in many industrial applicationsincluding as chemical intermediates, as processing aids, and asfunctional components in numerous formulations. Examples of fatty aminesinclude those produced in the present host cells and derived from fattyaldehyde precursors as described herein. The fatty amines and/or fattyamine compositions or blends that are produced herein can be used,individually or in suitable combinations or blends. The fatty amines ofthe present disclosure find use in industrial applications including,but not limited to, detergents (cleaners, thickeners, fabric softeners);dishwashing liquids; foaming- and wetting agents; demulsifiers(pharmaceuticals, paper, petroleum); emulsifiers (solvents, solventcleaners, silicones, oil, wax polish, leather treatment, triglycerides);surfactants; shampoos and conditioners; antistatic agents in the textileand plastics industry (textiles, polymers, electronics, electrostaticsprays, paper); fuel additives; lubricants and lubricant additives(grease thickeners, engine oil); paint thickeners; mineral processing;paper manufacture; petroleum production and refining (petroleumadditives, oil field chemicals); asphalt emulsifiers; corrosioninhibitors (acid, water treatment, metal workings, petroleum); gasoline-and fuel oil additives; flotation agents; epoxy curing agents; andagricultural chemicals and herbicides. In some aspects, the disclosurepertains to a method of producing a fatty amine composition includingfatty amines that are made of either a mixture of carbon chains or aspecific chain length that ranges from about C8 to about C24. In oneparticular aspect, the disclosure pertains to a method of producing afatty amine composition encompassing primary fatty amines (RNH₂). Inanother aspect, secondary fatty amines (R₂NH) and/or tertiary fattyamines (trialkyl (R₃N), dialkylmethyl (R₂NCH₃), and/or alkyldimethyl(RN(CH₃)₂)) are also contemplated. In another aspect, the presentdisclosure encompasses the production of primary amines that can becomethe primary building blocks for many industrial products as well asprovide the source material for numerous chemical derivatives such aspolyamines, ethoxylated amines, ethoxylated diamines, propoxylatedamines, amine salts, amine oxides, amides, ethoxylated amides, andnitriles. In related aspects, the method encompasses a geneticallyengineered production host suitable for making fatty amines and fattyamine compositions including, but not limited to, primary amines thatare suitable for producing chemical derivatives and compositions thereofincluding, but not limited to, polyamines, ethoxylated amines,ethoxylated diamines, propoxylated amines, amine salts, amine oxides,amides, ethoxylated amides, and nitriles.

In general, the fatty amine or fatty amine composition of the presentdisclosure is isolated from the extracellular environment of the hostcell. In some embodiments, the fatty amine or fatty amine composition isspontaneously secreted, partially or completely, from the host cell. Inalternative embodiments, the fatty amine or fatty amine composition istransported into the extracellular environment, optionally with the aidof one or more transport proteins. In still other embodiments, the fattyamine or fatty amine composition is passively transported into theextracellular environment.

The methods can produce fatty amines including a C8-C24 fatty amine. Insome embodiments, the fatty amine includes a C8, C9, C10, C11, C12, C13,C14, C15, C16, C17, C18, C19, C20, C21, C22, C23 and/or C24 fatty amine.In other embodiments, the fatty amine composition includes one or moreof a C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, and a C18fatty amine. In still other embodiments, the fatty amine compositionincludes C12, C14, C16 and C18 fatty amines; C12, C14 and C16 fattyamines; C14, C16 and C18 fatty amines; or C12 and C14 fatty amines. TheR group of a fatty amine, can be a straight chain or a branched chain.Branched chains may have more than one point of branching and mayinclude cyclic branches. In some embodiments, the branched fatty amineis a C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, C19,C20, C21, C22, C23 or C24 branched fatty amine. The R group of abranched or unbranched fatty amine can be saturated or unsaturated. Ifunsaturated, the R group can have one or more than one point ofunsaturation. In some embodiments, the unsaturated fatty amine is amonounsaturated fatty amine. In certain embodiments, the unsaturatedfatty amine is a C6:1, C7:1, C8:1, C9:1, C10:1, C11:1, C12:1, C13:1,C14:1, C15:1, C16:1, C17:1, C18:1, C19:1, C20:1, C21:1, C22:1, C23:1 ora C24:1 unsaturated fatty amine. In certain embodiments, the unsaturatedfatty amine is a C10:1, C12:1, C14:1, C16:1, or C18:1 unsaturated fattyamine. In other embodiments, the unsaturated fatty amine is unsaturatedat the omega-7 position. In certain embodiments, the unsaturated fattyamine has a cis double bond.

In one preferred embodiment, the fatty amine is a primary amineincluding, but not limited to, octyl amine, decyl amine, dodecyl amine,tetradecyl amine, hexadecyl amine, octadecyl amine, stearyl amine, andoleyl amine. In another embodiment, the fatty amine is a secondaryamine, for example, 1-dodecylamine (laurylamine), 1-hexadecylamine(palmitylamine), 1-octadecylamine (stearylamine), and the like. Inanother embodiment, the fatty amine is a tertiary amine, for example,1-octadecen-9-ylamine (oleylamine), and the like. Other examples offatty amines are alkyl dimethyl amines including, but not limited to,octyl dimethyl amine, decyl dimethyl amine, dodecyl dimethyl amine,tetradecyl dimethyl amine, hexadecyl dimethyl amine, octadecyl dimethylamine, and oleyl dimethyl amine. Still other examples of fatty aminesare dialkyl methyl amines including, but not limited to, dioctyl methylamine, didecyl methyl amine, didodecyl methyl amine, ditetradecyl methylamine, dihexadecyl methyl amine, and dioctadecyl methyl amine. Thedisclosure further contemplates fatty amines produced by the recombinanthost cells as described herein that can be used for chemicalderivatives, such as fatty amides (e.g., stearamide, oleamide,erucamide); quaternaries (e.g., tetramethyl ammonium chloride,tetramethyl ammonium bromide, tetraethyl ammonium bromide, tetrapropylammonium bromide); and ethoxylates (e.g., lauryl amine, stearyl amine,oleyl amine, octadecyl amine).

In other embodiments, the fatty amine includes a straight-chain fattyamine. In other embodiments, the fatty amine includes a branched-chainfatty amine. In yet other embodiments, the fatty amine comprises acyclic moiety. In some embodiments, the fatty amine is an unsaturatedfatty amine. In other embodiments, the fatty amine is a monounsaturatedfatty amine. In yet other embodiments, the fatty amine is a saturatedfatty amine. In another aspect, the disclosure features a fatty aminesproduced by any of the methods or any of the microorganisms describedherein, or a surfactant encompassing a fatty amine produced by any ofthe methods or any of the microorganisms described herein. In someembodiments, the fatty amine has a δ¹³C of about −15.4 or greater. Incertain embodiments, the fatty amine has a δ¹³C of about −15.4 to about−10.9, or of about −13.92 to about −13.84. In some embodiments, thefatty amines has an f_(M) ¹⁴C of at least about 1.003. In certainembodiments, the fatty amine has an f_(M) ¹⁴C of at least about 1.01 orat least about 1.5. In some embodiments, the fatty amine has an f_(M)¹⁴C of about 1.111 to about 1.124.

EXAMPLES

The following specific examples are intended to illustrate thedisclosure and should not be construed as limiting the scope of theclaims.

Example 1

Fatty aldehyde precursors and corresponding fatty amines were generatedin vivo by co-expressing a thioesterase ('tesA) and a carboxylic acidreductase (CarB) with a putrescine aminotransferase (ygjG) along withsupplementation of a nitrogen source (glutamate). The fatty aldehydeprecursors were converted into the corresponding amines.

The ygjG gene was cloned from the E. coli MG1655 strain via PCR with thefollowing primers:

Forward primer: (SEQ ID NO: 1)′5-AGGAGGAATAACATATGAACAGGTTACCTTCGAGCGCATCGGC-3′. Reverse primer:(SEQ ID NO: 2) ′5-CCCAAGCTTCGAATTCTTACGCTTCTTCGACACTTACTCGCATGGC C-3′.

The ygjG gene was then ligated into the expression vector pACYC (i.e.,high copy expression vector), to generate the plasmid pACYC-ygjG. Asecond plasmid was generated and named pCL1920-CarB-18-cTesA2-13C05(i.e., low copy plasmid), which contained the Mycobacterium smegmatiscarB gene and a variant of the thioesterase gene ('tesA) from E. coli.The two plasmids were co-transformed into an E. coli strain that doesnot produce fatty amines (DVD2.1, containing ΔfadE ΔtonA fabB-A329VP_(T5)-entD from the E. coli MG1655 strain) giving strain F16-YG. Thehost cells were also transformed with each of the plasmids separatelyfor use as controls giving control strains F16(pCL1920-CarB-18-cTesA2-13C05) and control strain YG (pACYC-ygjG).

The cells were grown at 32° C. in M9 minimal medium supplemented with 3%(w/v) glucose, 0.5% (v/v) TRITON X-100, 0.1 M bis-tris, pH 7.0, andinduced at OD₆₀₀˜1.0 with 1 mM isopropyl β-D-1-thiogalactopyranoside(IPTG). (IPTG triggers transcription of the lac operon, and is used toinduce protein expression where the gene is under the control of the lacoperator.) At the time of induction, 5 g/L L-glutamate was also added asa source of nitrogen. Strains containing pACYC-ygjG were grown in thepresence of the antibiotic carbenicillin, and strains containingpCL1920-CarB-18-cTesA2-13C05 were grown in the presence ofspectinomycin, in order to select for the respective plasmids. Afterovernight growth, the cultures of the three strains were supplementedwith an additional 10 g/L glucose and 5 g/L L-glutamate. Aliquots of 1mL of culture were frozen at 24 hours post-induction.

In order to prepare samples for analysis, 0.5 mL of ethyl acetate wereadded to each aliquot of culture. The samples were then vortexed atmaximum speed for 15 minutes and centrifuged for 5 minutes. The organicphase was analyzed with a Gas Chromatograph Mass Spectrometry (GCMS fromAgilent 6890) in EI mode (i.e., method: alkane 1 splitless CTC). TheF16-YG strain, in which ygjG, carB, and 'tesA were exogenouslyexpressed, yielded a unique peak at 7.6 minutes (FIG. 1 middle panel)that was not observed in either of the YG or F16 negative controls (FIG.1 top and bottom panel). The unique peak in the sample from the F16-YGculture at 7.6 minutes was identified as 1-dodecylamine by the NIST 05chemical compound library. An analytical reference standard purchasedfrom Sigma/Aldrich (Product #325163) was run back to back with theF16-YG sample, which confirmed the identity of the compound as1-dodecylamine by its retention time (FIG. 2) and by its ionfragmentation pattern (FIG. 3) showing characteristic fragments atm/z=142, 156 and 170 and a molecular ion at 185.

Example 2

Fatty aldehyde precursors and corresponding fatty amines can begenerated in vivo by co-expressing an acyl-ACP reductase (AAR) with aputrescine aminotransferase (ygjG) along with supplementation of anitrogen source (glutamate). The fatty aldehyde precursors can beconverted into the corresponding amines.

The ygjG gene can be cloned from the E. coli MG1655 strain via PCR withthe following primers:

Forward primer: (SEQ ID NO: 1)′5-AGGAGGAATAACATATGAACAGGTTACCTTCGAGCGCATCGGC-3′. Reverse primer:(SEQ ID NO: 2) ′5-CCCAAGCTTCGAATTCTTACGCTTCTTCGACACTTACTCGCATGGC C-3′.

The ygjG gene can then then be ligated into the expression vector pACYC,to generate the plasmid pACYC-ygjG. A second plasmid can be generatedand named pCL1920-aar, which is a pCL-based plasmid containing a genefor AAR from Synechococcus elongatus PCC7942 (aar). The two plasmids canbe co-transformed into the E. coli strain that does not produce fattyamines (supra) giving strain F17-YG. The host cells can also betransformed with each of the plasmids separately for use as controlsgiving control strain F17 (pCL1920-aar) and control strain YG(pACYC-ygjG).

The cells can be grown at 32° C. in M9 minimal medium supplemented with3% (w/v) glucose, 0.5% (v/v) TRITON X-100, 0.1 M bis-tris, pH 7.0, andinduced at OD₆₀₀˜1.0 with 1 mM isopropyl β-D-1-thiogalactopyranoside(IPTG). At the time of induction, 5 g/L L-glutamate can be also added asa source of nitrogen. Strains containing pACYC-ygjG can be grown in thepresence of the antibiotic carbenicillin (0.05 mg/mL), and strainscontaining pCL1920-aar can be grown in the presence of 0.1 mg/mLspectinomycin in order to select for the respective plasmids. Afterovernight growth, the cultures of the three strains can be supplementedwith an additional 10 g/L glucose and 5 g/L L-glutamate. Aliquots of 1mL of culture can be frozen at 24 hours post-induction.

In order to prepare samples for analysis, 0.5 mL of ethyl acetate can beadded to each aliquot of culture. The samples can then be vortexed atmaximum speed for about 15 minutes and centrifuged for about 5 minutesas needed. The organic phase can be analyzed with a Gas ChromatographMass Spectrometry (GCMS from Agilent 6890) in EI mode (i.e., method:alkane 1 splitless CTC). The F17-YG strain, in which ygjG and/or aar areexogenously expressed, are expected to yield one or more unique peaksthat represent fatty amines similar to what was observed in Example 1(supra) and that were not observed in either of the YG or F17 negativecontrols. Any unique peaks in the sample from the F17-YG culture can beidentified as fatty amines via the NIST 05 chemical compound library.For comparison, an analytical reference standard from Sigma/Aldrich(Product #325163) can be run back to back with the F17-YG sample, inorder to confirm the identity of the compound being produced by itsretention time and by its ion fragmentation pattern.

Example 3

Fatty aldehyde precursors and corresponding fatty amines can begenerated in vivo by co-expressing a thioesterase (TesA) and acarboxylic acid reductase (CarB) with a GABA aminotransferase (PuuE)along with supplementation of a nitrogen source (glutamate). The fattyaldehyde precursors can be converted into the corresponding amines. ThepuuE gene can be cloned from an E. coli strain via PCR with a suitableforward and reverse primer (similarly as taught in Examples 1 and 2,supra). The puuE gene can then be ligated into an expression vector(e.g., pACYC, supra), to generate the plasmid pACYC-puuE. A secondplasmid can be generated and named pCL1920-CarB-18-cTesA2-13C05, whichis a second expression vector containing the Mycobacterium smegmatiscarB gene and a variant of the thioesterase gene ('tesA) from E. coli(see Example 1, supra). The two plasmids can be co-transformed into theE. coli strain that does not produce fatty amines (supra) giving strainF18-PU. The host cells can also be transformed with each of the plasmidsseparately for use as controls giving control strain F18(pCL1920-CarB-18-cTesA2-13C05) and control strain PU (pACYC-puuE).

The cells can be grown at 32° C. in M9 minimal medium supplemented with3% (w/v) glucose, 0.5% (v/v) TRITON X-100, 0.1 M bis-tris, pH 7.0, andinduced at OD₆₀₀˜1.0 with 1 mM isopropyl β-D-1-thiogalactopyranoside(IPTG). At the time of induction, 5 g/L L-glutamate can be added as asource of nitrogen. Strains containing pACYC-puuE can be grown in thepresence of the antibiotic carbenicillin (0.05 mg/mL), and strainscontaining pCL1920-CarB-18-cTesA2-13C05 can be grown in the presence of0.1 mg/mL spectinomycin in order to select for the respective plasmids.After overnight growth, the cultures of the three strains can besupplemented with an additional 10 g/L glucose and 5 g/L L-glutamate.Aliquots of 1 mL of culture can be frozen at 24 hours post-induction.

In order to prepare samples for analysis, 0.5 mL of ethyl acetate can beadded to each aliquot of culture. The samples can then be vortexed atmaximum speed for about 15 minutes and centrifuged for 5 minutes asneeded. The organic phase can be analyzed with a Gas Chromatograph MassSpectrometry (GCMS from Agilent 6890) in EI mode (i.e., method: alkane 1splitless CTC). The F18-PU strain, in which puuE, carB, and/or 'tesA areexogenously expressed, are expected to produce one or more unique peaksthat are not observed in either of the PU or F18 negative controls. Theexpected unique peak in the sample from the F18-PU culture can then beidentified via the NIST 05 chemical compound library. An analyticalreference standard from Sigma/Aldrich (Product #325163) can be run backto back with the F18-PU sample, in order to confirm the identity of anovel fatty amine compound.

Example 4

Fatty aldehyde precursors and corresponding fatty amines can begenerated in vivo by co-expressing an AAR with a GABA aminotransferase(PuuE) along with supplementation of a nitrogen source (glutamate). Thefatty aldehyde precursors can be converted into the correspondingamines.

The puuE gene can be cloned from an E. coli strain via PCR with asuitable forward and reverse primer (similarly as taught in Example 1,supra). The puuE gene can be ligated into an expression vector (e.g.,pACYC, supra), to generate the plasmid pACYC-puuE. A second plasmid canbe generated and named pCL1920-aar, which is another expression vectorcontaining the gene for AAR from Synechococcus elongatus PCC7942 (aar).The two plasmids can be co-transformed into the E. coli strain that doesnot produce fatty amines (supra) giving strain F19-PU. The host cellscan also be transformed with each of the plasmids separately for use ascontrols giving control strain F19 (pCL1920-aar) and control strain PU(pACYC-puuE).

The cells can be grown at 32° C. in M9 minimal medium supplemented with3% (w/v) glucose, 0.5% (v/v) TRITON X-100, 0.1 M bis-tris, pH 7.0, andinduced at OD₆₀₀˜1.0 with 1 mM isopropyl β-D-1-thiogalactopyranoside(IPTG). At the time of induction, 5 g/L L-glutamate can be added as asource of nitrogen. Strains containing pACYC-puuE can be grown in thepresence of the antibiotic carbenicillin (0.05 mg/mL) and strainscontaining pCL1920-aar can be grown in the presence of 0.1 mg/mLspectinomycin, in order to select for the respective plasmids. Afterovernight growth, the cultures of the three strains can be supplementedwith an additional 10 g/L glucose and 5 g/L L-glutamate. Aliquots of 1mL of culture can be frozen at 24 hours post-induction.

In order to prepare samples for analysis, 0.5 mL of ethyl acetate can beadded to each aliquot of culture. The samples can then be vortexed atmaximum speed for about 15 minutes and centrifuged for about 5 minutesas needed. The organic phase can be analyzed with a Gas ChromatographMass Spectrometry (GCMS from Agilent 6890) in EI mode (i.e., method:alkane 1 splitless CTC). The F19-PU strain, in which puuE, and/or aarare exogenously expressed, are expected to produce a one or more uniquepeaks that are not observed in either of the PU or F19 negativecontrols. The expected unique peak in the sample from the F19-PU culturecan then be identified via the NIST 05 chemical compound library. Ananalytical reference standard from Sigma/Aldrich (Product #325163) canbe run back to back with the F19-PU sample, in order to confirm theidentity of a novel fatty amine compound.

Example 5

Fatty aldehyde precursors and corresponding fatty amines can begenerated in vivo by co-expressing a thioesterase (TesA) and acarboxylic acid reductase (CarB) with an amine dehydrogenase (e.g.,methylamine dehydrogenase of Paracoccus denitrificans or quinohemoprotein amine dehydrogenase of Pseudomonas spp.) along withsupplementation of a nitrogen source (ammonia). The fatty aldehydeprecursors can be converted into the corresponding amines.

The amine dehydrogenase (AD) gene from Paracoccus denitrificans orPseudomonas spp. can be cloned via PCR with a suitable forward andreverse primer (similarly as taught in Example 1, supra). The AD genecan then be ligated into an expression vector (e.g., pACYC, supra) togenerate the plasmid pACYC-AD. A second plasmid can be generated andnamed pCL1920-CarB-18-cTesA2-13C05, which is another expression vectorcontaining the Mycobacterium smegmatis carB gene and a variant of thethioesterase gene ('tesA) from E. coli (see Example 1, supra). The twoplasmids can be co-transformed into the E. coli strain that does notproduce fatty amines (supra) giving strain F20-AD. The host cells canalso be transformed with each of the plasmids separately for use ascontrols giving control strain F20 (pCL1920-CarB-18-cTesA2-13C05) andcontrol strain AD (pACYC-AD).

The cells can be grown at 32° C. in M9 minimal medium supplemented with3% (w/v) glucose, 0.5% (v/v) TRITON X-100, 0.1 M bis-tris, pH 7.0, andinduced at OD₆₀₀˜1.0 with 1 mM isopropyl β-D-1-thiogalactopyranoside(IPTG). At the time of induction, approximately 0.5-1 g/L ammonia can beadded as a source of nitrogen. Strains containing pACYC-AD can be grownin the presence of the antibiotic carbenicillin (0.05 mg/mL) and strainscontaining pCL1920-CarB-18-cTesA2-13C05 can be grown in the presence of0.1 mg/mL spectinomycin, in order to select for the respective plasmids.After overnight growth, the cultures of the three strains can besupplemented with an additional 10 g/L glucose and approximately 0.5-1g/L ammonia. Aliquots of 1 mL of culture can be frozen at 24 hourspost-induction.

In order to prepare samples for analysis, 0.5 mL of ethyl acetate can beadded to each aliquot of culture. The samples can then be vortexed atmaximum speed for about 15 minutes and centrifuged for 5 minutes asneeded. The organic phase can be analyzed with a Gas Chromatograph MassSpectrometry (GCMS from Agilent 6890) in EI mode (i.e., method: alkane 1splitless CTC). The F20-AD strain, in which AD, carB, and/or 'tesA areexogenously expressed, are expected to produce one or more unique peaksthat are not observed in either of the PU or F20 negative controls. Theexpected unique peak in the sample from the F20-AD culture can then beidentified via the NIST 05 chemical compound library. An analyticalreference standard from Sigma/Aldrich (Product #325163) can be run backto back with the F20-AD sample, in order to confirm the identity of anovel fatty amine compound.

Example 6

Fatty aldehyde precursors and corresponding fatty amines can begenerated in vivo by co-expressing an AAR with an amine dehydrogenase(e.g., methylamine dehydrogenase of Paracoccus denitrificans orquinohemo protein amine dehydrogenase of Pseudomonas spp.) along withsupplementation of a nitrogen source (ammonia). The fatty aldehydeprecursors can be converted into the corresponding amines.

The amine dehydrogenase (AD) gene can be cloned from an E. coli strainvia PCR with a suitable forward and reverse primer (similarly as taughtin Example 1, supra). The AD gene can be ligated into an expressionvector (e.g., pACYC, supra) to generate the plasmid pACYC-AD. A secondplasmid can be generated and named pCL1920-aar, which is another vectorcontaining the gene for AAR from Synechococcus elongatus PCC7942 (aar).The two plasmids can be co-transformed into the E. coli strain that doesnot produce fatty amines (supra) giving strain F21-AD. The host cellscan also be transformed with each of the plasmids separately for use ascontrols giving control strain F21 (pCL1920-aar) and control strain AD(pACYC-AD).

The cells can be grown at 32° C. in M9 minimal medium supplemented with3% (w/v) glucose, 0.5% (v/v) TRITON X-100, 0.1 M bis-tris, pH 7.0, andinduced at OD₆₀₀˜1.0 with 1 mM isopropyl β-D-1-thiogalactopyranoside(IPTG). At the time of induction, approximately 0.5-1 g/L ammonia can beadded as a source of nitrogen. Strains containing pACYC-AD can be grownin the presence of the antibiotic carbenicillin (0.05 mg/mL), andstrains containing pCL1920-aar can be grown in the presence of 0.1 mg/mLspectinomycin, in order to select for the respective plasmids. Afterovernight growth, the cultures of the three strains can be supplementedwith an additional 10 g/L glucose and approximately 0.5-1 g/L ammonia.Aliquots of 1 mL of culture can be frozen at 24 hours post-induction.

In order to prepare samples for analysis, 0.5 mL of ethyl acetate can beadded to each aliquot of culture. The samples can then be vortexed atmaximum speed for about 15 minutes and centrifuged for about 5 minutesas needed. The organic phase can be analyzed with a Gas ChromatographMass Spectrometry (GCMS from Agilent 6890) in EI mode (i.e., method:alkane 1 splitless CTC). The F21-AD strain, in which AD and/or aar areexogenously expressed, are expected to produce a one or more uniquepeaks that are not observed in either of the AD or F21 negativecontrols. The expected unique peak in the sample from the F21-AD culturecan then be identified via the NIST 05 chemical compound library. Ananalytical reference standard from Sigma/Aldrich (Product #325163) canbe run back to back with the F21-AD sample, in order to confirm theidentity of a novel fatty amine compound.

As is apparent to one with skill in the art, various modifications andvariations of the above aspects and embodiments can be made withoutdeparting from the spirit and scope of this disclosure. Suchmodifications and variations are within the scope of this disclosure.

We claim:
 1. A recombinant bacterial cell for production of a fattyamine, wherein the fatty amine has a carbon chain length of between 5 to24 carbons, comprising: (i) one or more expressed genes that encode anexogenous biosynthetic enzyme having thioesterase activity of EC number3.1.2.-, 3.1.1.5, or 3.1.2.14; (ii) one or more expressed genes thatencode an exogenous biosynthetic enzyme having carboxylic acid reductaseactivity of EC number 1.2.99.6; and (iii) one or more expressed genesthat encode an exogenous biosynthetic enzyme having aminotransferase (ECnumber 2.6.1) or amine dehydrogenase activity (EC number 1.4.9, 1.4.98,1.4.99), wherein said recombinant bacterial cell produces the fattyamine in vivo.
 2. The recombinant bacterial cell of claim 1, whereinsaid exogenous biosynthetic enzyme having thioesterase activity convertsan acyl-ACP or acyl-CoA to a fatty acid.
 3. The recombinant bacterialcell of claim 2, wherein said exogenous biosynthetic enzyme havingcarboxylic acid reductase activity converts said fatty acid to a fattyaldehyde.
 4. The recombinant bacterial cell of claim 3, wherein saidexogenous biosynthetic enzyme having aminotransferase or aminedehydrogenase activity converts said fatty aldehyde to a fatty amine. 5.The recombinant bacterial cell of claim 1, wherein said exogenousbiosynthetic enzyme having thioesterase activity is encoded by a tesAgene.
 6. The recombinant bacterial cell of claim 1, wherein saidexogenous biosynthetic enzyme having carboxylic acid reductase activityis encoded by a carB gene.
 7. The recombinant bacterial cell of claim 1,wherein said exogenous biosynthetic enzyme having aminotransferaseactivity is a putrescine or GABA aminotransferase.
 8. The recombinantbacterial cell of claim 7, wherein the putrescine aminotransferase isYgjG.
 9. The recombinant bacterial cell of claim 8, wherein said YgjG isencoded by an ygjG gene.
 10. The recombinant bacterial cell of claim 7,wherein the GABA aminotransferase is PuuE.
 11. The recombinant bacterialcell of claim 10, wherein said PuuE is encoded a puuE gene.
 12. Therecombinant bacterial cell of claim 1, wherein said exogenousbiosynthetic enzyme having amine dehydrogenase activity is a methylaminedehydrogenase.
 13. The recombinant bacterial cell of claim 12, whereinsaid methylamine dehydrogenase is from Paracoccus denitrificans.
 14. Therecombinant bacterial cell of claim 1, wherein said recombinantbacterial cell is selected from the group consisting of Escherichia,Bacillus, Cyanophyta, Lactobacillus, Zymomonas, Rhodococcus, andPseudomonas.
 15. The recombinant bacterial cell of claim 14, whereinsaid Escherichia is Escherichia coli.
 16. The recombinant bacterial cellof claim 14, wherein said Cyanophyta is selected from the groupconsisting of Prochlorococcus, Synechococcus, Synechocystis, Cyanothece,and Nostoc punctiforme.
 17. The recombinant bacterial cell of claim 14,wherein said Cyanophyta is selected from the group consisting ofSynechococcus elongatus PCC7942, Synechocystis sp. PCC6803, andSynechococcus sp. PCC7001.
 18. A cell culture comprising the recombinantbacterial cell of claim
 1. 19. A method of producing a fatty amine,comprising culturing the bacterial cell of claim 1 in a fermentationbroth containing a carbon source.
 20. The method of claim 19, furthercomprising harvesting fatty amines that collect in the fermentationbroth.